Magnesium phosphate hydrogels

ABSTRACT

A hydrogel comprising a colloidal suspension of MIXMIIYPZ two-dimensional nanocrystals in water, wherein MI is Na+ and/or Li+, MII is Mg2+ or a mixture of Mg2+ with one or more Ni2+, Zn2+, Cu2+, Fe2+ and/or Mn2+, P is a mixture of dibasic phosphate ions (HPO42−) and tribasic phosphate ions (PO43−). X ranges from about 0.43 to about 0.63, Y ranges from about 0.10 to about 0.18, Z ranges from about 0.29 to about 0.48, X, Y, Z being mole fractions, is provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/060,462, filed Jun. 8, 2018, which in turn is a U.S. national stageapplication of PCT/CA2016/051415 filed Dec. 2, 2016, which in turnclaims benefit, under 35 U.S.C. § 119(e), of U.S. provisionalapplication Ser. No. 62/265,570, filed on Dec. 10, 2015.

SEQUENCE LISTING

A sequence listing in electronic (ASCII text file) format is filed withthis application and incorporated herein by reference. The name of theASCII text file is “2020_2904_seq_listing.txt”; the file was created onJan. 20, 2021; the size of the file is 4 KB.

FIELD OF THE INVENTION

The present invention relates to magnesium phosphate hydrogels. Morespecifically, the present invention is concerned with such gels andtheir uses as scaffolds for bone tissue engineering, as drug deliverysystems and in pastes for cleaning dental implants.

BACKGROUND OF THE INVENTION Two-Dimensional (2D) Layered Materials

Over the past decade, the field of two-dimensional (2D) layeredmaterials has grown extensively, especially after the isolation andcharacterization of graphene. 2D nanomaterials have attracted a greatinterest since they present extraordinary properties that are usuallyabsent in their bulk form. Recent progress in 2D nanomaterialstechnologies also paved the way in developing advanced biomaterials, andin the large family of 2D nanomaterials exfoliated synthetic clays havebeen used in many advanced technological applications

The design of nanomaterials with a well-defined 2D morphology and theirlarge-scale manufacturing at low cost, in particular, remain crucialchallenges to unfold the very promising future of nanotechnology. Infact, the synthesis of 2D nanomaterials is often time-consuming andinvolves multi-step procedures that may use toxic and/or expensivechemicals for the exfoliation/delamination process, or hydrothermalprocess at high temperatures and pressures. Overall these methods mightbe expensive, do not offer scope for scalability, and are inappropriatefor the synthesis of biomaterials. In recent years, sonochemicaltechniques have been extensively used in the synthesis of nanostructuredmaterials. During the acoustic cavitation process, very hightemperatures (>5000 K), pressures (>20 MPa), and cooling rates (>10¹⁰K/s) can be achieved upon the collapse of the bubble. However, theapplication of sonochemical process to a large-scale level is a verycomplicated task.

Clays are plate-like polyions with a heterogeneous charge distributionthat forms a physical gel in water at concentrations higher than 40mg/mL due to the simultaneous presence of positive and negative chargesthat give rise to electrostatic and van der Waals interactions. Thisallows the gel to behave as a thixotropic material due to the formationof a 3D network of particles known as the “house of cards” structure.Thixotropic materials can be liquefied by applying mechanical energyallowing the physical gel to behave as a liquid; then when themechanical stress is removed Brownian motions drive the particles intocontact to reform the 3D network and the liquefied dispersion becomesgel-like again.

Inorganic Biomaterials, Phosphates, Magnesium

Several inorganic biomaterials such as calcium phosphates,hydroxyapatite, beta tri-calcium phosphates, monetite, brushite, andorthosilicic acid have been studied as osteoinducers. However, thesematerials present insufficient in vivo degradation which results in slowresorption. They also have limited injectability, and tissueregeneration limits making necessary the development of a newbiomaterial generation which can facilitate the formation of functionaltissues.

Magnesium is the fourth most common metal in human body, 50% of thebody's magnesium is stored in bone, and it shares many chemicalsimilarities with calcium. Magnesium plays an important role in mineralmetabolism promoting calcification, hydroxyapatite (HA) crystalformation, increases bone cell adhesion, proliferation, anddifferentiation. Among phosphate-based materials, magnesium phosphateshave demonstrated to be biocompatible and resorbable in vivo.

Binary transition metal phosphates because of their interestingindustrial properties have received considerable amount of attention.The synthesis of a series of phosphates M^(I)M^(II)PO₄.H₂O (M^(I)=K,NH₄; M^(II)=Mg, Mn, Fe, Co, Ni) was first reported in 1933.

Cleaning Dental Implants

Oral biofilm can accumulate onto the surface of dental implants causinginfection and compromising implant survival. The accumulation ofbacterial biofilm on titanium (Ti) implants changes the surfacebiocompatibility and initiates peri-implant diseases (peri-implantmucositis and peri-implantitis). These can cause marginal bone loss andeventually implant failure. Therefore, regular removal of oral biofilmfrom Ti implants is critical to maintain oral health and ensurelong-term implant success.

Home-use and professional oral hygiene techniques are thus highlyindicated to prevent or manage the peri-implant infections and thusincrease implant survival. Personal and professional plaque control withbrushes, polishing cups and pastes has indeed been used to removebiofilms covering implant surfaces. These techniques should be capableof removing bacterial biofilms without negatively affecting the implantbiocompatibility, but they currently cannot. Further, even though thesetechniques decrease the symptoms of peri-implant infections, they do notachieve complete biofilm removal from the implants. In fact, availableprophylaxis pastes and toothpastes present limited efficiency incleaning implant surfaces because they were all originally designed forcleaning teeth not implants. In particular, they are made of organicthickeners and surfactants that can bind to titanium and alter itsproperties.

Conventional toothpastes have indeed been developed to promote dentalhealth and assist the mechanical removal of biofilm from teeth withbrushes. The composition of most toothpastes includes abrasives(hydrated silica, calcium carbonate, alumina), surfactants (glycerin,sorbitol), organic thickeners (xanthan, cellulose gums), andantimicrobials (fluoride, triclosan). However, these additives can havea negative impact on the stability and chemical properties of implantsurfaces.

Fluoride ions can initiate surface corrosion of Ti metal and alloys,altering its surface chemistry, topography and roughness. The effect offluoride is not limited to the time of oral hygiene procedure becausethe fluoride could be retained and concentrated in the plaque, and itcan be found in saliva 24 hours after the use of fluoridated oralhygiene products.

Furthermore, organic macromolecules are known to spontaneously adsorb tometals causing alteration in their physical chemistry and surfacecharge. Natural and synthetic inorganic clays such as Laponite (layeredmagnesium silicate) are used in the prophylaxis and toothpastes asbinders or stabilizer, but they are commonly incorporated with otherorganic thickeners (i.e. xanthan gum) to obtain the optimal consistencyof a dentifrice. The organic compounds can attach tightly to the implantsurface which make it impossible to clean the surface without damagingits microtexture. Moreover, clays are silicate based gels that could betoo abrasive on implant surfaces.

In addition, the abrasives incorporated in regular toothpastes orpolishing pastes can damage implants surfaces and increase theirroughness. Abrasives are indeed added to enhance the cleaning action ofthe toothbrush and to physically scrub the external surface ofteeth/implants, removing the organic pellicle (salivary proteins),plaque bacteria and other extrinsic stains. Calcium carbonate, silicaand alumina are the common abrasive elements used in the current pastes.

Prophylaxis instruments, such as brushes or rubber cups, have been usedto decontaminate implants and remove the attached biofilms with orwithout using prophylaxis pastes. They showed a relative moderateefficiency in biofilm removal without negative effects on the implantsurfaces. However, implant surface damage was reported with the use ofhighly abrasive rubber cups and/or polishing paste.

In view of the above, it advisable to use toothpastes and instrumentswith low abrasiveness for daily oral hygiene maintenance for subjects'with Ti implants. In fact, toothpastes have to be carefully selectedwhen implant restorations are present. Unfortunately, no specific“implant-paste” exists. Colgate™ Total toothpaste is a representativeconventional toothpaste that is used for personal daily care mainly toreduce plaque and prevent gum infections. It composed of antimicrobials(sodium fluoride, triclosan), organic thickeners (cellulose gum andcopolymers), abrasives (hydrated silica and titanium dioxide), andhumectants (glycerin and sorbitol).

Bone Regeneration

Minimally invasive surgical interventions have been shown to reduceoperation and anesthesia time, minimize intra-operative complications,minimize postoperative pain, shorten recovery duration and hospital staywhich in turn reduce morbidity and mortality rates, and minimize thecost of the intervention. Thus, such interventions have gained greatdeal of publicity.

Bone regeneration procedures require invasive and painful interventions.Bone fixation for instance involve invasive incision through skin andmuscle to expose bone in order to place fixation plates. Suchintervention increase risk of damage to adjacent anatomical structuresuch as nerve injury.

Pain management in bone regeneration interventions is limited to the useof drugs such as non-steroidal anti-inflammatories, opioids,acetaminophen and local anesthetics. However, these drugs have severallimitations. Non-steroidal anti-inflammatories delay bone healing andincrease the risk of gastrointestinal diseases. Opioids are controlleddrugs, and have major side effects such as constipation and addiction.Acetaminophen is usually not effective in moderate or severe bone pain.Local anesthetics are relatively the most effective and have the leastside effects, however they are limited by their short duration ofaction.

SUMMARY OF THE INVENTION

In accordance with the present invention, there is provided a hydrogelcomprising a colloidal suspension of M^(I) _(X)M^(II) _(Y)P_(Z)two-dimensional nanocrystals in water, wherein:

M^(I) is Na⁺ and/or Li⁺,

M^(II) is Mg²⁺ or a mixture of Mg²⁺ with one or more Ni²⁺, Zn²⁺, Cu²⁺,Fe²⁺ and/or Mn²⁺,

P is a mixture of dibasic phosphate ions (HPO₄ ²⁻) and tribasicphosphate ions (P₄ ³⁻),

X ranges from about 0.43 to about 0.63,

Y ranges from about 0.10 to about 0.18, and

Z ranges from about 0.29 to about 0.48,

X, Y, Z being mole fractions.

There is also provided the above hydrogel, wherein X ranges from about0.45 to about 0.56, from about 0.45 to about 0.55, preferably from about0.45 to about 0.53, more preferably from about 0.50 to about 0.58, andmost preferably is about 0.52.

There is also provided any and all of the above hydrogels, wherein Yranges from about 0.13 to about 0.18, preferably from about 0.14 toabout 0.18, more preferably from about 0.13 to about 0.16, and mostpreferably is about 0.15.

There is also provided any and all of the above hydrogels, wherein Zranges from about 0.30 to about 0.39, preferably from about 0.31 toabout 0.37, more preferably from about 0.34 to about 0.37, and mostpreferably is about 0.33.

There is also provided any and all of the above hydrogels, wherein M^(I)is Na⁺; wherein M^(I) is Li⁺, or M^(I) is a mixture of Na⁺ and Li⁺.

There is also provided any and all of the above hydrogels, whereinM^(II) is Mg²⁺; M^(II) is a mixture of Mg²⁺, and one or more Ni²⁺, Zn²⁺,Cu²⁺, Fe²⁺ and/or Mn²⁺; or wherein M^(II) is a mixture of Mg²⁺ and Fe²⁺.

There is also provided any and all of the above hydrogels, comprisingone or more of Ni²⁺, Zn²⁺, Cu²⁺, Fe²⁺ and/or Mn²⁺ in a total molefraction of up to about 0.3Y, more preferably a total mole fraction ofup to about 0.2Y, and more preferably a total mole fraction of about0.16Y.

There is also provided any and all of the above hydrogels, wherein M^(I)is Na⁺, M^(II) is Mg²⁺, X is 0.516, Y is 0.144, and Z is 0.34; whereinM^(I) is Na⁺, M^(II) is Mg²⁺, X is 0.45, Y is 0.18, and Z is 0.37;wherein M^(I) is Na⁺, M^(II) is Mg²⁺, X is 0.53, Y is 0.13, and Z is0.34; wherein M^(I) is Na⁺, M^(II) is a mixture of Mg²⁺ and Fe²⁺, X is0.55, Y is 0.14, and Z is 0.31; wherein M^(I) is Na⁺, M^(II) is Mg²⁺, Xis 0.52, Y is 0.13, and Z is 0.35; wherein M^(I) is Na⁺, M^(II) is Mg²⁺,X is 0.55, Y is 0.14, and Z is 0.31, or wherein M^(I) is Na⁺, M^(II) isMg²⁺, X is 0.56, Y is 0.13, and Z is 0.31.

There is also provided any and all of the above hydrogels, having a pHbetween about 7 and about 11, between about 7 to about 10, preferablybetween about 7 and about 9, more preferably pH between about 7.5 andabout 8.5, yet more preferably between about 7.5 and about 8, and morepreferably a pH of about 7.8.

There is also provided any and all of the above hydrogels, comprisingbetween about 5% and about 50%, preferably between about 5% and about25%, more preferably between about 5% and about 15%, and most preferablyabout 10% by weight of M^(I) _(X)M^(II) _(Y)P_(Z), based on the totalweight of the gel.

There is also provided any and all of the above hydrogels, comprisingbetween about 50% and about 95%, preferably between about 75% and about95%, more preferably between about 85% and about 95%, most preferablyabout 90% of water by weight based on the total weight of the gel.

There is also provided any and all of the above hydrogels, comprising upto 15%, preferably up to about 10%, more preferably between about 4 andabout 9% of hydration water by weight based on the total weight of thegel.

There is also provided any and all of the above hydrogels, wherein thehydrogel comprises M^(I) _(X)M^(II) _(Y)P_(Z) two-dimensionalnanocrystals agglomerated and forming interconnected planes with waterin empty spaces between the agglomerated nanocrystals.

There is also provided any and all of the above hydrogels, wherein thehydrogel comprises a honeycomb network of extended sheet-likeface-to-face aggregates that are bent, twisted, branched, andintertangled with few edge-to-face contacts

There is also provided any and all of the above hydrogels, furthercomprising one or more additive.

There is also provided any and all of the above hydrogels, furthercomprising one or more bioactive agents.

There is also provided any and all of the above hydrogels, for use inbone tissue engineering.

There is also provided any and all of the above hydrogels, for use as ascaffold for bone tissue engineering.

There is also provided any and all of the above hydrogels, for promotingbone regeneration and/or peri-implant bone growth

There is also provided any and all of the above hydrogels, for use as adrug delivery system.

In another related aspect of the invention, there is provided a scaffoldfor bone growth, for bone repair, and/or for bone regenerationcomprising any of the above hydrogels.

In another related aspect of the invention, there is provided a bonegraft and/or a bone regeneration material comprising any of the abovehydrogels.

In another related aspect of the invention, there is provided a methodfor:

-   -   promoting bone regeneration,    -   promoting bone growth (for example peri-implant bone growth),    -   treating a bone defect, and/or    -   treating a bone injury,

the method comprising the step of administering any of the abovehydrogels at a site of need.

There is also provided the above method, wherein the administering stepcomprises implanting the hydrogel or injecting the hydrogel. There isalso provided the above method, wherein the site of need is a bonedefect or a bone injury.

In another related aspect of the invention, there is provided a kitcomprising a container containing any of the above hydrogels andinstructions for using the hydrogel for promoting bone regeneration,promoting bone growth (for example peri-implant bone growth), treating abone defect, and/or treating a bone injury. There is also provided theabove kit, wherein the container is a syringe.

In another related aspect of the invention, there is provided apharmaceutical composition comprising one or more bioactive agents andany of the above hydrogels as a carrier for the bioactive agent. Thereis also provided the above pharmaceutical composition, wherein thepharmaceutical composition is an implant or an injectable. There is alsoprovided the above pharmaceutical composition, wherein the bioactiveagent is a local anesthetic.

In another related aspect of the invention, there is provided a methodof delivering a bioactive agent to a patient, the method comprising thestep of administering any of the pharmaceutical composition to thepatient. In another related aspect of the invention, there is provided amethod of targeting delivery of a bioactive agent to a site of need of apatient, the method comprising the steps of administering any of thepharmaceutical composition to the site of need. There is also providedthe above methods, wherein the site of need is a bone defect or a boneinjury. There is also provided the above methods, wherein saidadministering step comprises implanting the hydrogel or injecting thehydrogel.

In another related aspect of the invention, there is provided a pastefor cleaning dental implant, the paste comprising any of the abovehydrogels mixed with an abrasive agent.

There is also provided the above paste, wherein the gel has a pH betweenabout 9 and about 10.

There is also provided the above paste, wherein, in the hydrogel, M^(I)is Na⁺, M^(II) is Mg²⁺, X is 0.56, Y is 0.13, and Z is 0.31.

There is also provided the above paste, wherein the abrasive agent is asilica, such as a magnesium phosphate silica, a nano-silicate or calciumcarbonate.

There is also provided the above paste, wherein the abrasive agent ishydrated silica nanoparticles.

There is also provided the above paste, wherein abrasive agent particleshave a particles size up to about 500 nm, preferably up to about 400 nm,and more preferably ranging from about 200 to about 300 nm.

There is also provided the above paste, comprising from about 5 to about60%, preferably from about 20 to about 40%, more preferably about 30% byweight of the abrasive agent, based on the total weight of the paste.

There is also provided the above paste, further comprising one or moreadditives.

In another related aspect of the invention, there is provided a methodof manufacturing any of the above hydrogel, the method comprisingproviding a first reservoir containing a first aqueous solutioncomprising Mg²⁺ ions, dibasic phosphate ions (HPO₄ ²⁻) and tribasicphosphate ions (PO₄ ³⁻), and optionally further comprising one or moreNi²⁺, Zn²⁺, Cu²⁺, Fe²⁺ and/or Mn²⁺,

-   -   providing a second reservoir containing a second aqueous        solution comprising Na⁺ and/or Li⁺ ions,    -   providing a small-volume mixing chamber flowably connected to        said first and second reservoir and having an outlet,    -   simultaneously feeding said first and second solutions to the        mixing chamber, thereby manufacturing said hydrogel, and    -   collecting the hydrogel via the outlet of the mixing chamber.

BRIEF DESCRIPTION OF THE DRAWINGS

In the appended drawings:

FIG. 1 shows an apparatus for manufacturing the hydrogel describedherein:

FIG. 2 shows the total points used to determine the different crystalphases of the ternary diagram of the system NaOH—Mg(OH)₂—H₃PO₄;

FIG. 3 shows the ternary diagram of the Mg(OH)₂—NaOH—H₃PO₄ system withthe different phases obtained by mixing the three components atdifferent mole fractions;

FIG. 4 shows the X-ray diffraction pattern of a new unidentifiedcrystalline phase obtained in the are a labelled “New crystalline phaseand mixed Mg/PO₄ phases in FIG. 3;

FIG. 5 shows the thermogravimetric analysis of the differentformulations: from a to d—Formulations A, B, C, and D, respectively;

FIG. 6 shows the pH of the colloidal suspension as a function of thereaction time;

FIG. 7 is a picture of the suspension after 30 seconds from thebeginning of the reaction;

FIG. 8 shows the NMP colloidal suspension after 10 minutes;

FIGS. 9 A and B show the NMP nanocrystals evolution during the reaction;

FIG. 10 shows the evolution of G′, G″ and δ of the gel of formulation Aas a function of the increasing shear stress with time;

FIG. 11 shows the evolution of G′, G″ and δ of different gelformulations as a function of the increasing shear stress withtime—rheology measurements of formulation A;

FIG. 12 shows the evolution of G′, G″ and δ of different gelformulations as a function of the increasing shear stress withtime—rheology measurements of formulation B;

FIG. 13 shows the evolution of G′, G″ and δ of different gelformulations as a function of the increasing shear stress withtime—rheology measurements of formulation C;

FIG. 14 shows the evolution of G′, G″ and δ of different gelformulations as a function of the increasing shear stress withtime—rheology measurements of formulation D;

FIG. 15 shows the physical aspect of the NMP suspension (A) in asyringe, (B) while injected through an insulin needle (160 μm internaldiameter), and (C) after injection;

FIG. 16 is a representative TEM micrograph of a freeze-fracturedcarbon-platinum replica of a 5% w/w NMP suspension;

FIG. 17 is a high magnification TEM micrograph of the carbon-platinumreplica grid showing the laminar structure of the ultra-thinnanocrystals of formulation A with a face-to-face arrangement and athickness of 4-7 nm;

FIG. 18 is a TEM micrograph of the NMP colloidal suspensions ofFormulation A;

FIG. 19 is a TEM micrograph of the NMP colloidal suspensions ofFormulation B;

FIG. 20 is a TEM micrograph of the NMP colloidal suspensions ofFormulation C;

FIG. 21 is a TEM micrograph of the NMP colloidal suspensions ofFormulation D;

FIG. 22 shows the XRD patterns of different powders showing the partialor total conversion of nanocrystalline NMP into crystalline Newberyite;

FIG. 23 is a TEM micrograph of a NMP colloidal dispersion of formulationA with a concentration of 1% v/v in water (In the inset, selected areaelectron diffraction (SAED) shows the nanocrystallinity of the NMPnanocrystals.);

FIG. 24 is a Titan Krios micrograph of the NMP gel of formulation Ashowing the very thin structure of the 2D nano-sheet;

FIG. 25 shows the stability of the thixotropic suspension over time as afunction of the ratio [Na]/([Na]+[K];

FIG. 26 shows the physical aspect after one week of NMP suspensions withdifferent ratios of [Na]/([Na]+[K]);

FIG. 27 shows the ternary diagram of the pH as a function of the molefraction of Mg(OH)₂, NaOH, and H₃PO₄;

FIG. 28 shows vials tubes with the colloidal dispersions (a) after thesynthesis and (b) after 3 days—the gel synthesized using LiOH (on theleft in both pictures) remained stable while the gel using KOH (on theright) lost its stability and converted into Newberyite (MgHPO₄.3H₂O);

FIG. 29 shows the FT-IR spectrum of the dried and washed NMP powder offormulation A;

FIG. 30 shows the FT-IR spectrum of the same powder after calcination at700° C. for 8 hours;

FIG. 31 shows the NMR spectra of formulation A, taken using a14Tspectrometer;

FIG. 32 shows the NMR spectra of the biomaterial of formulation A afterimmersion in D₂O;

FIG. 33 shows a) XPS depth profile experiment of the NMP colloidalsuspension synthesized on formulation A, b) the deconvolution of highresolution XPS spectra of P2p confirmed the presence of PO₄ ³⁻ and HPO₄²⁻, and c) the variation of the at. % of Na⁺ and Mg²⁺ of formulation Aafter mild etching using Ar ions;

FIGS. 34 A and B show the deposition of NMP on a negatively chargedglass surface;

FIGS. 35 A and B show NMP powder deposited on a positively charged glasssurface;

FIG. 36 shows the results of the metabolic activity using Alamar-Blueassay and live/dead assay on HF cells—number of HF cells;

FIG. 37 shows the results of the metabolic activity using Alamar-Blueassay and live/dead assay on HF cells—percentage of Living HF cells;

FIG. 38 shows the results of Live-Dead assay of formulation A at day 1.a-c, Channel splitting for the different dyes used (CalceinAM/Etd-1/Hoechst 33258); d, Micrograph after merging the three channels.The scale bar length is 100 μm;

FIG. 39. shows the results of Live-Dead assay of formulation B at day 1.a-c, Channel splitting for the different dyes used (CalceinAM/Etd-1/Hoechst 33258). d, Micrograph after merging the three channels.The scale bar length is 100 m;

FIG. 40. shows the results of Live-Dead assay of formulation A at day 4.a-c, Channel splitting for the different dyes used (CalceinAM/Etd-1/Hoechst 33258). d, Micrograph after merging the three channels.The scale bar length is 50 m;

FIG. 41. shows the results of Live-Dead assay of formulation B at day 4.a-c, Channel splitting for the different dyes used (CalceinAM/Etd-1/Hoechst 33258). d, Micrograph after merging the three channels.The scale bar length is 50 m;

FIG. 42 is a SEM micrograph showing the adhesion and colonization ofosteoblast cells onto NMP nanocrystals;

FIG. 43 shows the mRNA quantitative expression of ALP of mouse bonemarrow cells grown for 21 days on, from left to right, Newberyite(MgHPO₄.3H₂O) (normalized values), NMP formulation B, and Cattiite(Mg₃(PO₄)₂.22H₂O);

FIG. 44 shows the mRNA quantitative expression of OCN of mouse bonemarrow cells grown for 21 days on, from left to right, Newberyite(MgHPO₄.3H₂O) (normalized values), NMP formulation B, and Cattiite(Mg₃(PO₄)₂.22H₂O);

FIG. 45 shows the mRNA quantitative expression of OPN of mouse bonemarrow cells grown for 21 days on, from left to right, Newberyite(MgHPO₄.3H₂O) (normalized values), NMP formulation B, and Cattiite(Mg₃(PO₄)₂.22H₂O);

FIG. 46 shows the mRNA quantitative expression of COL1A1 of mouse bonemarrow cells grown for 21 days on, from left to right, Newberyite(MgHPO₄.3H₂O) (normalized values), NMP formulation B, and Cattiite(Mg₃(PO₄)₂.22H₂O);

FIG. 47 shows the mRNA quantitative expression of RunX2 of mouse bonemarrow cells grown for 21 days on, from left to right, Newberyite(MgHPO₄.3H₂O) (normalized values), NMP formulation B, and Cattiite(Mg₃(PO₄)₂.22H₂O);

FIG. 48 shows μ-CT 3D models of the bone defects at day 3, 7 and 14;

FIG. 49 shows histology and histomorphometry analysis (14 days aftersurgery): maison trichrome stain (collagen), ALP stain (osteoblasts) andTRAP stain (osteoclasts) in the control and the NMP-treated defects;

FIG. 50 shows the percentage of bone-implant-contact (BIC) in thecontrol and the NMP-treated defects;

FIG. 51 shows the percentage of collagen in the control and theNMP-treated defects (Maison trichrome stain);

FIG. 52 shows the number of osteoblasts (ALP stain) in the control andthe NMP-treated defects;

FIG. 53 shows the number of osteoclasts (TRAP stain) in the control andthe NMP-treated defects;

FIG. 54 shows μ-CT 3-D models and coronal histological sections ofTi-implants showing more bone (lighter in color in μ-CT and darker inhistology) in contact with implant in NMP-coated implants;

FIG. 55 is a FIB image showing bone matrix undergoing mineralization byosteoblasts in NMP-treated defect at day 7;

FIG. 56 is a FIB image showing collagen fibers undergoing mineralizationin NMP-treated defect at day 7;

FIG. 57 shows the results of qRT-PCR showing that the expression ofRunX2 was up-regulated in NMP treated, at day 3 (on the left) comparedto the control, however, no significant difference was observed at day14 (on the right);

FIG. 58 shows the results of qRT-PCR showing that the expression ofCOL1A1 was up-regulated in NMP treated, at day 3 (on the left) comparedto the control, however, no significant difference was observed at day14 (on the right);

FIG. 59 shows A) (a-c) photographs of a rotary brush loaded with the NMPgel, the developed implant-paste and Colgate toothpaste and (d-f)photographs of the Eppendorf tubes containing the NMP gel, implant-pasteand Colgate toothpaste respectively and B) a representative TEMmicrograph of a freeze-fractured carbon-platinum replica of a 10% w/wNMP suspension showing the 3D structure and interactions of thenanocrystals composing the NMP gel;

FIG. 60 shows X-ray Photoelectron Spectroscopy (XPS) surveys (A), a barchart (B), scanning Electron Microscope images at a magnification of×10,000 (C) and photographs (D) illustrating the cleaning effect ofrotary prophylaxis brush at different brushing time on the elementalcomposition and topography of biofilm-contaminated Ti surfaces;

FIG. 61 shows Scanning Electron Microscope images (magnification×10,000, top row) and photographs (bottom row) showing the topography ofthe biofilm-contaminated Ti surfaces after brushing with the NMP gel,the gel containing different concentrations of hydrated silica andColgate toothpaste (brushing time was 1 minute);

FIG. 62 shows XPS surveys (A) and a bar chart (B) comparing the cleaningefficiency of the NMP gel and the gel containing differentconcentrations of hydrated silica (Brushing time was 1 minute);

FIG. 63 shows XPS surveys (A) and a bar chart (B) showing the change inthe elemental composition of uncontaminated Ti surfaces after cleaningthem with the rotary brush and optimized implant-paste (NMP gelcontaining 30% hydrated silica) and a commercial toothpaste (Colgate),brushing time was 1 minute;

FIG. 64 shows bar charts (A) and confocal laser scanning microscopeimages (B), comparing the surface roughness of polished Ti surfacesafter cleaning with the prophylaxis brush, the optimized implant-paste(NMP gel containing 30% hydrated silica) and commercial toothpaste(Brushing time is 1 minute);

FIG. 65 shows XPS surveys (A) and a bar chart (B) comparing the cleaningefficacy of the prophylaxis brush, the optimized implant-paste andColgate toothpaste (brushing time was 1 minute);

FIG. 66 shows bar charts (A) and live dead staining (fluorescence)images (B) comparing the bacterial removal efficiency of the prophylaxisbrush, the optimized implant-paste and Colgate toothpaste (brushing timewas 1 minute);

FIG. 67 shows the drug release in vitro showing that the gel can controlthe liberation of the local anesthetic (loading of NMP withmepivacaine);

FIG. 68 is the Korsmeyer-Peppa's fitting for the cumulative drugreleased from gel+mepivacaine samples;

FIG. 69 shows the UV-Vis spectra of the mepivacaine released from thegel after 24 hours; and

FIG. 70 show the results of the radiant heat test used to evaluate theheat tolerance of mice in vivo using the mouse-hindpaw-model; theseresults showed that the NMP loaded with mepivacaine provides analgesiaand the analgesic action of mepivacaine was prolonged by NMP;

FIG. 71 shows A) weight bearing test results and B) guarding testresults for saline, mepivacaine, NMP, and NMP+mepivacaine treatment;

FIG. 72 shows micro-CT sagittal, coronal sections and 3 Dreconstructions showing bone formation at fracture site after saline,mepivacaine, NMP, and NMP+mepivacaine treatment; and

FIG. 73 shows 3-points pending test results after saline, mepivacaine,NMP, and NMP+mepivacaine treatment.

DETAILED DESCRIPTION OF THE INVENTION Hydrogel

In accordance with the present invention, there is provided a hydrogelcomprising a colloidal suspension of M^(I) _(X)M^(II) _(Y)P_(Z)two-dimensional nanocrystals in water.

In the above chemical formula:

M^(I) is a monovalent cation and is Na⁺ and/or Li⁺,

M^(II) is a divalent cation and is Mg²⁺ or a mixture of Mg²⁺ with one ormore Ni²⁺, Zn²⁺, Cu²⁺, Fe²⁺ and/or Mn²⁺,

P is a mixture of dibasic phosphate ions (HPO₄ ²⁻) and tribasicphosphate ions (PO₄ ³⁻),

X ranges from about 0.43 to about 0.63,

Y ranges from about 0.10 to about 0.18, and

Z ranges from about 0.29 to about 0.48,

X, Y, Z being mole fractions.

It will be readily apparent to the skilled person that, since X, Y and Zare mole fractions, their sum should be 1 (give or take the roundingerrors). This is indeed the standard definition of mole fraction in theart: “In chemistry, the mole fraction is defined as the amount of aconstituent divided by the total amount of all constituents in amixture. The sum of all the mole fractions is equal to 1”. Herein, themole fractions calculation takes only the divalent cations (M^(II)),phosphate anions (P) and monovalent cations (M^(I)) into account. Waterand optional additives that can be added to the gel are not considered.

In embodiments, X is 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50,0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, or0.62 or more. In these or other embodiments, X is 0.63, 0.62, 0.61,0.60, 0.59, 0.58, 0.57, 0.56, 0.55, 0.54, 0.53, 0.52, 0.51, 0.50, 0.49,0.48, 0.47, 0.46, 0.45, or 0.44 or less. In embodiments, X is about anyof the preceding values.

In embodiments, Y is 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, or 0.17or more. In these or other embodiments, Y is 0.18, 0.17, 0.16, 0.15,0.14, 0.13, 0.12, or 0.11 or less. In embodiments, Y is about any of thepreceding values.

In embodiments, Z is 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36,0.37, 0.38, 0.39, 0.40, 0.41, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, or0.47 or more. In these or other embodiments, Z is 0.48, 0.47, 0.46,0.45, 0.44, 0.43, 0.42, 0.41, 0.40, 0.39, 0.38, 0.37, 0.36, 0.35, 0.34,0.33, 0.32, 0.31, or 0.30 or less. In embodiments, Z is about any of thepreceding values.

In preferred embodiments, X ranges from about 0.45 to about 0.56, fromabout 0.45 to about 0.55, preferably from about 0.45 to about 0.53, morepreferably from about 0.50 to about 0.58, and most preferably is about0.52.

In preferred embodiments, Y ranges from about 0.13 to about 0.18,preferably from about 0.14 to about 0.18, more preferably from about0.13 to about 0.16, and most preferably is about 0.15.

In preferred embodiments, Z ranges from about 0.30 to about 0.39,preferably from about 0.31 to about 0.37, more preferably from about0.34 to about 0.37, and most preferably is about 0.33.

In preferred embodiments, the monovalent cation (M^(I)) is Na⁺.

In embodiments, the monovalent cation (M^(I)) is Li⁺.

In embodiments, the monovalent cation (M^(I)) is a mixture of Li⁺ andNa⁺.

In preferred embodiments, the divalent cation (M^(II)) is magnesium(Mg²⁺) only.

In other embodiments, part of the magnesium is replaced by one or moreof Ni²⁺, Zn²⁺, Cu²⁺, Fe²⁺, and/or Mn²⁺. In other words, M^(II) is amixture of Mg²⁺, with one or more of Ni²⁺, Zn²⁺, Cu²⁺, Fe²⁺ and/or Mn²⁺,or any combination or subset thereof. In embodiments, the one or more ofNi²⁺, Zn²⁺, Cu²⁺, Fe²⁺ and/or Mn²⁺, is Fe²⁺. In gels comprising suchmixtures, the one or more of Ni²⁺, Zn²⁺, Cu²⁺, Fe²⁺ and/or Mn²⁺, may bepresent in a total mole fraction of up to about 0.3Y (which means thatMg²⁺ is present in a mole fraction of at least about 0.7Y). In preferredembodiments, the gel comprises the one or more of Ni²⁺, Zn²⁺, Cu²⁺, Fe²⁺and/or Mn²⁺ in a total mole fraction of up to about 0.2Y. Inembodiments, the gels comprise the one or more of Ni²⁺, Zn²⁺, Cu²⁺, Fe²⁺and/or Mn²⁺, in a total mole fraction of about 0.16Y (and thus Mg²⁺ inpresent in a mole fraction of about 0.84Y).

It will be apparent to the skilled person that the ratio of dibasicphosphate ions (HPO₄ ²⁻) to tribasic phosphate ions (PO₄ ³⁻) in the gelwill depend on the exact pH of the gel.

In embodiments, the hydrogel has a pH between about 7 to about 11,preferably between about 7 to about 10, between about 7 and about 9,more preferably between about 7.5 and about 8.5, yet more preferablybetween about 7.5 and 8, most preferably a pH of about 7.8. Non-limitingexamples of hydrogels with (more or most) preferred pH include:

Hydrogels from the Examples below X Y Z pH Example 3  0.516  0.144 0.347.8 (M^(I) = Na⁺, M^(II) = Mg²⁺) Example 1, Formulation B 0.45 0.18 0.377.8 (M^(I) = Na⁺, M^(II) = Mg²⁺) Example 4 0.53 0.13 0.34  7.95 (M^(I) =Na⁺, M^(II) = Mg²⁺) Example 2 0.55 0.14 0.31 8.1 (M^(I) = Na⁺, M^(II) =Mg²⁺ + Fe²⁺) (0.02 Fe + 0.12 Mg) Example 1, Formulation A 0.52 0.13 0.358.3 (M^(I) = Na⁺, M^(II) = Mg²⁺) Example 2 0.55 0.14 0.31  8.46 (M^(I) =Na⁺, M^(II) = Mg²⁺) Example 3 0.56 0.13 0.31 9.6 (M^(I) = Na⁺, M^(II) =Mg²⁺)

Thus, in preferred embodiments,

-   -   M^(I) is Na⁺, M^(II) is Mg²⁺, X is 0.516, Y is 0.144, and Z is        0.34;    -   M^(I) is Na⁺, M^(II) is Mg²⁺, X is 0.45, Y is 0.18, and Z is        0.37;    -   M^(I) is Na⁺, M^(II) is Mg²⁺, X is 0.53, Y is 0.43, and Z is        0.34;    -   M^(I) is Na⁺, M^(II) is a mixture of Mg²⁺ and Fe²⁺, X is 0.55, Y        is 0.14 (0.02 Fe+0.12 Mg), and Z is 0.31;    -   M^(I) is Na⁺, M^(II) is Mg²⁺, X is 0.52, Y is 0.13, and Z is        0.35;    -   M^(I) is Na⁺, M^(II) is Mg²⁺, X is 0.55, Y is 0.14, and Z is        0.31; and/or    -   M^(I) is Na⁺, M^(II) is Mg²⁺, X is 0.56, Y is 0.13, and Z is        0.31.

In any and all of the above hydrogels, the amount of M^(I) _(X)M^(II)_(Y)P_(Z) in the gel typically ranges between about 5% and about 50% byweight based on the total weight of the gel, for example between about5% and about 25%, or between about 5% and about 15%. In preferredembodiments, the gel comprises about 10% of M^(I) _(X)M^(II) _(Y)P_(Z).

In any and all of the above hydrogels, the amount of water (as adispersing phase) in the gel typically ranges between about 50% andabout 95% by weight based on the total weight of the gel, for examplebetween about 75% and about 95%, or between about 85% and about 95%. Inpreferred embodiments, the gel comprises about 90% of water. Adistinction should be drawn between water as a dispersing phase andhydration water. Water as a dispersing phase is the medium in which thenanosheets are dispersed. This water can be removed by drying the gel ata relatively low temperature, for example a temperature below theboiling temperature of water, such as 80° C. This process will produce aproduct that looks and feels dry, but that still contain hydrationwater.

On the other hand, hydration water consists in molecules of water thatare bonded or somehow associated with a solid (for example entrappedwithin it). These molecules are typically only removed from the solid byheating the solid above the boiling temperature of water, often wellabove this temperature, for example between 100 and 250° C. The abovehydrogel typically contains hydration water. For example, it may containup to about 15% of hydration water by weight based on the total weightof the gel, for example up to about 10%, or between about 4 and about9%.

When observed by transmission electron microscopy (TEM), in embodiments,the gel morphology comprises thin nano-plates or nanosheets (M^(I)_(X)M^(II) _(Y)P_(Z) two-dimensional nanocrystals). More specifically,these nanosheets can be about 200 nm wide, very thin (e.g. about 10 nmthick) and up to 1 m long. As seen by TEM, these nanosheets agglomerate,and form interconnected planes (see for example FIGS. 18 to 21).

Herein, a “colloidal suspension” refers to a mixture comprisingmicroscopically dispersed insoluble particles (herein the M^(I)_(X)M^(II) _(Y)P_(Z) two-dimensional nanocrystals) suspended throughouta medium (herein water), in which the particles do not settle or take along time to settle appreciably.

Herein, “nanocrystals” are crystalline particles having at least onedimension smaller than 100 nanometers. Herein, “two-dimensionalnanocrystals” (2D nanocrystals) are thin sheet-like nanocrystals. Inother words, the thickness of the 2D nanocrystals is much smaller thantheir width and length. In embodiments of the invention, the M^(I)_(X)M^(II) _(Y)P_(Z) 2D nanocrystals that are up to about 10 nm thick.For example, their thickness may range between about 4 and about 7 nm.The length of the nanocrystals can be as high as about 1 m, for example600 nm, and their width can be as high as about 250 nm, for example 200nm. (See for example FIG. 24).

The hydrogel of the invention takes the form of a colloidal suspensionof two-dimensional nanocrystals. In embodiments, the 2D nanocrystalsform bundles or aggregates that together produce a 3D network, with thewater composing the medium of the hydrogel in the empty spaces betweenthe bundled nanocrystals. More specifically, the nanocrystals maypartially overlap each other resulting in a honeycomb network ofextended sheet-like face-to-face aggregates that are bent, twisted,branched, and intertangled with generally few edge-to-face contacts.

Herein, the terms “agglomerate”, “aggregate” and “bundle” are usedinterchangeably.

In embodiments of any and all of the above hydrogels, the gel can alsocomprise one or more additives, such as nanoparticles (for example ofsilica), alginate, chitosan, or polyethylene glycol.

In embodiments of any and all of the above hydrogels, the gel can alsocomprise one or more bioactive agents, depending of the desiredproperties and its end use. Such agents will be discussed below.

Methods of Manufacturing the Hydrogel

In another aspect, the present invention provides methods ofmanufacturing the above hydrogel.

In these methods, the various ions can be provided using any of theirwater-soluble salts, oxides, acids or bases, which will typically beprovided as aqueous solutions. For biological application,pharmaceutically acceptable starting materials are preferred. Inparticular, the starting materials shown in the following table can beused.

TABLE 1 Starting Materials (preffered starting materials are in bold)Ions Starting Material Na⁺ NaOH, Na₂HPO₄, Na₅P₃O₁₀, NaH₂PO₄ Li⁺ LiOHMg²⁺

, MgCl₂, MgO, Mg(H₂PO₄)₂ and Mg₃(PO₄)₂ Ni²⁺ NiCl₂, Ni(CH₃COO)₂ Zn²⁺ZnCl₂, Zn(CH₃COO)₂, Zn(OH)_(2,) ZnO Cu²⁺ CuCl₂, Cu(OH)₂ Fe²⁺ FeCl₂, Mn²⁺MnCl₂, PO₄ ³⁻ and PO₄ ³⁻

, Na₂HPO₄, Na₅P₃O₁₀, NaH₂PO₄

Of note, in the above, a given starting material can provide two typesof ions at once.

The hydrogel of the invention can be prepared by mixing togethersolutions of the above starting materials. In preferred embodiments, asolution of the starting materials for the sodium and/or lithium ions isadded to a solution containing the other starting materials.

Such simple mixing is adequate for producing small volume batches (forexample 50 mL). However, for larger volume batches, the solution may notbe homogenized quickly enough to produce the hydrogel and other phaseswill rather undesirably be obtained.

To produce larger volumes of gel, it is advantageous to use a continuousmethod (rather than a batch method). More specifically, there isprovided a method of manufacture the above hydrogel, in which smallvolumes of the solutions are mixed, preferably continuously mixed, toproduce the hydrogel.

This can be accomplished by:

-   -   providing a first reservoir containing a first aqueous solution        comprising Mg²⁺ ions (alone or as a mixture Mg²⁺ with one or        more Ni²⁺, Zn²⁺, Cu²⁺, Fe²⁺ and/or Mn²⁺), dibasic phosphate ions        (HPO₄ ²⁻) and tribasic phosphate ions (PO₄ ³⁻),    -   providing a second reservoir containing a second aqueous        solution comprising Na⁺ and/or Li⁺ ions,    -   providing a small-volume mixing chamber flowably connected to        said first and second reservoir and having an outlet,    -   simultaneously feeding said first and second solutions to the        mixing chamber, thereby manufacturing said hydrogel, and    -   collecting the hydrogel via the outlet of the mixing chamber.

In this method, the small-volume mixing chamber is of a volumesufficiently small to allow rapid and homogeneously mixing of bothsolutions. In embodiments, the mixing chamber has a volume of up toabout 100 ml, for example up to about 50 ml, up to about 25 ml.

Small volumes of both solutions should be mixed with a sufficientturbulence to form the hydrogel. In embodiments, a turbulent regime witha Reynolds number >4000 will allow adequate mixing of the solutions andcontinuous production of the hydrogel. The turbulence may be controlledby adjusting the flow velocity of both solutions and the morphology(volume and shape) of the mixing chamber.

In embodiments, the mixing chamber can be provided with a stirrer.

FIG. 1 shows an embodiment of an apparatus allowing implementing theabove method.

Properties of the Hydrogel

In embodiments, the hydrogel may present one or more of the followingproperties/advantages.

The hydrogels have a controlled pH that makes them suitable forbiological applications.

The hydrogels present long term stability.

The hydrogels are thixotropic.

The hydrogels are injectable (through high gauge needles).

The hydrogels are biocompatible.

The hydrogels are bioresorbable.

The hydrogels control the release of bioactive agents.

The hydrogels can trigger unique osteogenic activities. The hydrogelscan accelerate bone healing and/or osseointegration by enhancingcollagen formation, osteoblasts differentiation and/or osteoclastsproliferation through up-regulation of COL1A1, RunX2, ALP, OCN and/orOPN.

Use as Scaffolds for Bone Tissue Engineering

Because, in embodiments, the present hydrogels can be injected throughhigh gauge needles into bone defects, can accelerate bone healing andosseointegration (The results reported below in the Examples show asignificant enhancement of bone healing and osseointegration compared toa control group, and a total resorption after only two weeks) and arebioresorbable, they could bring a paradigm shift in the fields ofminimally invasive orthopedic and craniofacial interventions. Indeed,they could minimize the invasiveness of such interventions. Thehydrogels could potentially replace conventionally used cements and(bio)ceramics.

The hydrogels as described above can thus be used in bone tissueengineering, notably to promote bone regeneration and peri-implant bonegrowth. They provide a temporary support media as well as a resorbablegraft, the hydrogels being eventually replaced by bone.

Therefore, in a related aspect of the invention, there is provided ascaffold for bone growth, for bone repair, and/or for bone regenerationcomprising the above hydrogel.

There is also provided a bone graft or bone regeneration materialcomprising the above hydrogel.

There are also provided methods for:

-   -   promoting bone regeneration,    -   promoting bone growth (in embodiments, peri-implant bone        growth),    -   treating a bone defect, and/or    -   treating a bone injury,        these methods comprising the steps of administering the hydrogel        at a site of need. In embodiments, said administering step        comprises implanting the hydrogel. In other embodiments, said        administering step comprises injecting the hydrogel. In        embodiments, the site of need is a bone defect or a bone injury.

The term “bone defect” as used herein includes, but is not limited to,defects or voids/gaps resulting from compression fractures, benign bonecysts, diseased bone, high energy trauma, peri-articular fractures,cranial-maxillo facial fractures, osteoporotic reinforcement (i.e. screwaugmentation), joint arthrodesis, joint arthroplasty and periodontalreconstruction.

There is also provided a kit comprising a container containing thehydrogel and instructions for using the hydrogel for promoting boneregeneration, promoting bone growth (in embodiments, peri-implant bonegrowth), treating a bone defect, and/or treating a bone injury asdescribed. In embodiments, the container is a syringe.

In the above, the hydrogel can optionally comprise one or more additivesand/or bioactive agents. The additives may be those discussed above. Thebioactive agents will be discussed in the next section.

Use as a Drug Delivery System

The hydrogels as described above can be used as drug delivery systems.

Therefore, in a related aspect of the invention, there is provided apharmaceutical composition comprising one or more bioactive agents andthe hydrogel (as described above, for example including variousadditives) as a carrier for the bioactive agent. In preferredembodiments, the pharmaceutical composition is an implant or aninjectable.

There is also provided a method of delivering a bioactive agent to apatient, the method comprising the step of administering the abovepharmaceutical composition to the patient.

There is also provided a method of targeting delivery of a bioactiveagent to a site of need of a patient, the method comprising the steps ofadministering the pharmaceutical composition to the site of need. Inembodiments, the site of need is a bone defect or a bone injury.

In embodiments of the above methods, said administering step comprisesimplanting the hydrogel. In other embodiments of the above methods, saidadministering step comprises injecting the hydrogel.

The bioactive agents carried by the above hydrogels can be any suchagent known in the art. Neutral and alkaline bioactive agents aregenerally preferred. Acidic bioactive agents can also be used. Someacidic bioactive agents, if they lower too much the pH of the gel, mayhowever destabilize the hydrogel. In many cases, these agents cannevertheless be used as destabilization can be avoided by using a morealkaline gel, which will result in a product with in a final pH in thestability range of the hydrogel. An example of gel with a bioactiveagent is provided in Example 4.

Non-limiting examples of bioactive agents that can be carried by theabove hydrogels include local anesthetics such as mepivacaine,antibiotics such as imipenem, and beta blockers such as propranolol, aswell as those discussed in the next paragraph.

In preferred embodiments, the hydrogel is used for bone tissueengineering as described above and as a drug delivery systemsimultaneously. In other words, in the scaffold for bone growth, thebone graft material, the bone regeneration material, the methods and thekit described in the previous section, the hydrogel comprises abioactive agent for delivery to the patient. Suitable bioactive agentswhen the hydrogel is used in bone tissue engineering as described aboveinclude anesthetics, antibiotics, hormones and growth factors (i.e.osteogenic, vasogenic, or neurogenic growth factors) and proteins (i.e.osteopontin). Preferred bioactive agents in such case includeanesthetics, more preferably local anesthetics, as well as antibioticsand osteogenic proteins. Examples of local anesthetics includemepivacaine. Examples of antibiotics include imipenem. Examples ofhormones include melatonin. Examples of growth factors include plateletderived growth factors (PDGF), transforming growth factors (TGF-β),insulin-like growth factors (IGF's), fibroblast growth factors (FGF's),epidermal growth factor (EGF), human endothelial cell growth factor(ECGF), granulocyte macrophage colony stimulating factor (GM-CSF), nervegrowth factor (NGF), vascular endothelial growth factor (VEGF),cartilage derived morphogenetic protein (CDMP). Examples of osteogenicproteins include OP-1, OP-2, BMP2, BMP3, BMP4, BMP9, DPP, Vg-1, 60 A,and Vgr-1, including naturally sourced and recombinant derivatives ofthe foregoing.

In such embodiments, the hydrogels advantageously provide pain reliefand a minimally invasive technique for bone repair. Indeed, a materialthat can relief pain and be administered through minimal invasiveprocedures (e.g. injection) could bring a paradigm shift to the fieldsof orthopedic and craniofacial interventions, for example. This wouldpotentially minimize the invasiveness of bone regeneration procedures,shorten the healing period and mobilization time, while eliminating orreducing the need for systemic drugs administration for pain management.

In embodiments, the hydrogel controls (for example, retards or extends)the delivery of the bioactive agent, thereby potentially enhancing itstherapeutic window. This is notably the case with local anaestheticmepivacaine (see the Example below).

Use in a Paste for Cleaning Dental Implants

The hydrogels as described above can also be used to produce a paste forcleaning dental implants.

Compared to conventional toothpastes commonly used for daily personalcare, the paste of the invention is specifically designed for cleaningdental implants, which have cleaning requirements that differsignificantly from natural teeth. To the inventor's knowledge, there iscurrently no product on the market specially designed and optimized forimplant surface decontamination.

In embodiments, the paste of the invention allows removing biofilmcontamination from titanium implant surfaces, while minimizingtopographical changes to these surfaces (i.e. without affecting surfaceintegrity). In contrast, regular commercial toothpastes, which areorganic-based, are less effective in that context and may evencontaminate the titanium implant surfaces—see the Examples below.

The paste of the invention could allow dentists and patients to removebiofilm from implants, control the peri-implant infections and/or favorre-osseointegration in case of bone loss. It could also be used forsurgical decontamination of implant surfaces or professional cleaning ofimplants during maintenance visits. It could also be used for dailypersonal care to clean titanium abutments in case of overdenture or evento clean exposed implant surfaces. Indeed, when wearing dental implants,titanium surfaces just below the crown, i.e. the “neck” of the implant,are commonly exposed.

Therefore, in an aspect of the invention, there is provided a paste forcleaning dental implants comprising the above hydrogel mixed with anabrasive agent. In the paste for cleaning dental implants, the hydrogelacts as a thickener and as carrier for the abrasive agent.

In preferred embodiments, the pH of the gel is between about 9 and about10, especially is the implants to be cleans are made of titanium. Onesuch gel is a gel in which, M^(I) is Na⁺, M^(II) is Mg²⁺, X is 0.56, Yis 0.13, and Z is 0.31.

In preferred embodiments, the paste is completely inorganic, i.e. it isfree of organic compounds.

Turning to the abrasive agent, hard abrasive materials with largeparticle sizes should preferably be avoided as they can induce surfacesscratches or rounded edges on the implant, thus potentially increasingplaque accumulation. As such, the abrasive agent should have arelatively small average particle size, for example up to about 500 nm,preferably up to about 400 nm, and more preferably from about 200 toabout 300 nm. Suitable abrasive agents include particles of silica,including magnesium phosphates silica, nano-silicates (that showosteoconductive properties that help inducing and accelerating boneregeneration) and/or calcium carbonate. More preferably, the abrasiveagent is hydrated silica nanoparticles, especially those with averageparticles size of about 200 to about 300 nm.

The abrasive agent can be present in the paste at a concentrationranging from about 5% to about 60%, preferably from about 20% to about40%, and more preferably about 30% by weight based on the total weightof the paste.

The paste for cleaning dental implants can comprise further additives,in particular such additives that are known as useful in dental cleaningpastes. Such additives include taste enhancers, coloring agents,sparkles as well as other functional ingredients. As noted above, suchadditives should be carefully selected to avoid inducing contaminationof the implants with organic compounds.

Definitions

Herein, “to implant” means to insert something into a person's body, forexample (but not limited to) by surgery. An “implant” is a material thatis intended/designed to be implanted into a person's body.

Herein, “to inject” means to introduce something into a person's bodyusing a needle. An “injectable” is a material that is intended/designedto be injected into a person's body.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext.

The terms “comprising”, “having”, “including”, and “containing” are tobe construed as open-ended terms (i.e., meaning “including, but notlimited to”) unless otherwise noted.

Any and all combinations and sub-combinations of the embodiments andfeatures disclosed herein are encompassed by the present invention. Forexample, all the disclosed components, properties and uses of the gelmay be combined.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. All subsets of values within the ranges arealso incorporated into the specification as if they were individuallyrecited herein.

Similarly, herein a general chemical structure with various substituentsand various radicals enumerated for these substituents is intended toserve as a shorthand method of referring individually to each and everymolecule obtained by the combination of any of the radicals for any ofthe substituents. Each individual molecule is incorporated into thespecification as if it were individually recited herein. Further, allsubsets of molecules within the general chemical structures are alsoincorporated into the specification as if they were individually recitedherein.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed.

No language in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Herein, the term “about” has its ordinary meaning. In embodiments, itmay mean plus or minus 10% or plus or minus 5% of the numerical valuequalified.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

Other objects, advantages and features of the present invention willbecome more apparent upon reading of the following non-restrictivedescription of specific embodiments thereof, given by way of exampleonly with reference to the accompanying drawings.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention is illustrated in further details by the followingnon-limiting examples.

Example 1—Magnesium Phosphate Gels that Up-Regulate Bone Formation andBone Regeneration

Here, we describe a novel nanocrystalline material with a 2Dnanostructure and relevant properties for biomedical applications. Wewere able to synthesize a 2D biomaterial with properties such asbiocompatibility, bioresorption, long term stability, thixotropy and/orinjectability using a simple, potentially scalable, method. Wediscovered that sodium ions can regulate the precipitation of magnesiumphosphate by interacting with the surface of the crystals causing apreferential crystal growth resulting in 2D morphology. The 2D materialgave rise to a physical hydrogel base on a nanocrystalline material.This hydrogel was characterized in vitro and in vivo. We show below thatit has a combination of osteogenic activities and accelerates bonehealing and osseointegration by enhancing collagen formation,osteoblasts differentiation and osteoclasts proliferation throughup-regulation of COL1A1, RunX2, ALP, OCN and OPN.

Experimental Section

Study and Characterization of the NaOH—Mg(OH)—H₃PO₄ System

The title ternary system was investigated by varying the mole fractionof NaOH, Mg(OH)₂, and H₃PO₄ in different solutions. A fixed volume of 7mL was used in all chemical reactions and the maximum reagentsconcentration was 10.5 mmol, in order to avoid any possibleconcentration effect. The ternary diagram was built using 141 differentpoints obtained by mixing the three components at different molefractions (FIG. 2). Precipitates obtained during the determination ofthe ternary diagram were prepared using the following procedure. 85 mgof Mg(OH)₂ were dissolved in 2.2 mL of H₃PO₄ 1.5 M and after completedissolution 3.8 mL of NaOH 1.5 M were added under vigorous stirring.After mixing the two solutions, the resulting colloidal suspension waslet stand for 2 hours, centrifuged at 4000 rpm for 5 minutes, and thesupernatant was discarded. The solid precipitate was vacuum dried atroom temperature and stored for characterization. The different crystalphases of the precipitates obtained during the ternary diagram wereidentified by means of X-ray diffraction (XRD). The diffraction patternsof the dried precipitates were recorded with a Bruker D8 Discover(Bruker AXS GmbH, Karlsruhe, Germany) from 50 to 58θ 2θ with a coppersource (λ_(Cu,Ka)=1.5406 Å) at 40 kV and 40 mA and GADDS detector. Thediffraction patterns were processed with EVA software (Bruker AXS GmbH,Karlsruhe, Germany) and phase composition was determined by comparingthe acquired spectra with the phases identified in the InternationalCentre for Diffraction Data (ICDD) database PDF-4.

Composition of the Stable NMP (NaMgPhosphate) Suspension

The elemental composition of stable NMP suspensions was determined usingInductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) witha Thermo Scientific iCAP 6000 Series ICP-OES (Thermo Fisher ScientificInc, East Grinstead, UK). In a typical procedure, 6 mg of dried NMPpowder was digested for 2 hours at 95° C. in 5 mL of HNO₃ 67% tracemetals basis and all samples were prepared in triplicate. Afterdigestion, the samples were let to cool down at room temperature andthen diluted with deionized water up to 50 mL. From this solution 1 mLwas taken and diluted with deionized water to 10 mL and measured byICP-OES. The calibration curves were prepared using freshly preparedstandards solution of Mg²⁺, Na⁺, and P₄ ³⁻ with a concentration of 10,5, 2, 1, and 0.1 ppm in HNO₃ 4%. The standard solutions were prepared bydilution from a certified standard solution of 1000 ppm in HNO₃ 4% (SCPScience Inc, Baie D′Urf{tilde over (e)}, Canada). The analysis pump ratewas set to 50 rpm, the plasma radio frequency power was 1150 W, theauxiliary and nebulizer gas flow were set to 0.5 L min⁻¹. Allmeasurements were performed in axial/radius mode.

Fourier Transform Infrared Spectroscopy (FT-IR) of the dried and heattreated NMP precipitates were recorded using a Perkin Elmer Spectrum Two(Perkin Elmer Inc, Waltham, Mass., USA) with single bounce diamond forAttenuated Total Reflectance (ATR). Spectra were recorded at roomtemperature from 450 to 4000 cm⁻¹ with a resolution of 4 cm⁻¹ and 64scans.

Thermogravimetric analysis (TGA) was performed to calculate the amountof crystallization water of the dried NMP precipitates (SDT Q600 TAInstruments, TA Instruments-Waters L.L.C. New Castle, USA). TGA was donein vertical mode on a platinum pan from 30 to 1100° C. using a heatingrate of 5° C./min, and in air atmosphere with a purge flow rate of 100mL min-.

In a typical synthesis to produce NMP, in a solution of H₃PO₄(ηH₃PO₄=0.37) the pH value increased from 0.8 to 1.9 after adding anddissolving Mg(OH)₂ (ηMg(OH)₂=0.18). The addition of the NaOH solution(ηNaOH=0.45, pH 8.3) provoked the instantaneous formation of a whiteliquid suspension made of nanocrystals with a uniform size of 50 nm. ThepH of the suspension remained constant for 4 minutes before it began toslowly decrease and the white suspension became grey and solidified.During the following 30 minutes, the pH of the suspension stabilized at7.8, and the small nanocrystals increased their size forming the finalsuspension composed of 2D nanocrystals with an undulate structure (FIGS.7 to 9). The observed pH acidification during the reaction might arisefrom deprotonation of acidic phosphate moieties during the reaction as aresult of the formation of tribasic orthophosphate (PO₄ ³⁻).

Rheology, Nanocrystals Morphology, and 3-D Structure of the NMPColloidal Suspension

Colloidal suspensions of NMP were subjected to rheological measurementsto determine the shear stress required for the gel-liquid transition,the liquid-gel transition time (t_(L-G)) upon removal of the shearstress, and the viscosity. The rheological experiments were performedwith a rheometer AR2000 of TA Instruments (TA Instruments-Waters L.L.C.New Castle, USA) using parallel plates with a diameter of 40 mm and adistance between plates of 0.1 mm. Oscillatory measurements wereconducted at a constant frequency (f=1 Hz), and the oscillatory stresssweeps ranged from 1 to 700 Pa. The equilibration time before to run themeasurements was set to 10 minutes and all the measurements wereperformed at room temperature.

The force required to inject the NMP nanocrystals through an insulinneedle of 160 μm of internal diameter was measured using the instrumentMach-1 V500cs and Mach-1 Motion software version 4.3.1 (BiomomentumInc., Laval, Canada). The force was measured with a multiple-axis loadcell of 70 N (resolution of 0.007 N) and acquisition rate of 100 Hz. Thegel was loaded into the syringe avoiding the presence of bubble and thenthe plunger was inserted into the load cell. The force value wasmeasured applying a constant vertical stage velocity of 1 mm s⁻¹(resolution of 0.1 μm). The syringe loaded with deionized water requireda force of 0.14±0.01 N, while for the NMP gels of formulation A-D werecomprised in a range of 0.22±0.03 N and 0.77±0.04 N. At lower stagevelocity (0.3 mm s⁻¹) the required force to inject the NMP samples wasconsiderably higher reaching a maximum of 18±1 N and a minimum of 9±1.2N.

Freeze-fracture replica was used to investigate the organization andmorphology of the nanocrystals forming the thixotropic material. Thestable NMP colloidal suspension was quickly frozen in liquid nitrogen(cooling rate >10⁵ K s⁻¹) immobilizing the nanocrystals instantaneously(Acharya et al., Journal of International Oral Health 2014, 6, 36 1).The resulting frozen suspension was fractured, the ice was removed byvacuum freeze etching, and a thin layer of carbon was sputtered onto thesurface to produce a carbon replica. The sample surface was shadowedwith platinum vapor and the carbon-metal replica was put on aFormvar/Carbon coated copper mesh-200 grid (2SPI, Structure Probe Inc,West Chester, USA) and examined by Transmission Electron Microscopy(TEM) using a Tecnai T12 working at 120 kV (FEI Inc, Hillsboro, Oreg.,USA). Selected Area Electron Diffraction and TEM imaging of the waterdispersion of the NMP nanocrystals were performed on a TEM gridFormvar/Carbon coated copper mesh-200 grid (2SPI, Structure Probe Inc,West Chester, USA). The grid was prepared by deposition of a 5 μL dropof a 1% v/v water dispersion of the NMP gel, and the drop was blottedwith filter paper after 90 seconds. Scanning Electron Microscopy (SEM)was used to characterize the nanocrystals at different time pointsduring the reaction to obtain the NMP and the adhesion of osteoblastonto the surface of the nanocrystals. For time-point analysis, analiquot of 2 mL was withdrawn from the reaction and poured into aBuchner filter, washed with water first (20 mL), ethanol (20 mL), andthen dried in a vacuum oven at 25° C. This process was performed at 0.5,5, 10, and 30 minutes from the beginning of the reaction. SEM wascarried out using a FEI Inspect F-50 FE-SEM (FEI Inc, Hillsboro, Oreg.,USA) operated at 10 kV. For osteoblast adhesion, cultures were fixedusing a 2.5% glutaraldehyde solution for 20 minutes, and then dehydratedusing a series of ethanol solutions from 50 to 100%. Afterwards, thesamples were dried with ethanol/trichlorotrifluoroethane solutions usingthe subsequent ratios: 75/25, 50/50, 25/75, and 0/100 for 15 minutes and0/100 until complete evaporation.

Zeta potential measurements were carried out to assess the superficialcharge of the nanocrystals using a Malvern Nano ZS equipped withdisposable folded capillary cells (Malvern Instruments Ltd, Malvern,UK). The concentration of the gel used in the measurements was 20 mgmL⁻¹ and the temperature was kept constant at 25° C. To assess thesimultaneous presence of negative and positive charges, the interactionof the NMP nanocrystals with positively and negatively charged glasssurfaces was studied (FIGS. 34 and 35). NMP nanocrystals were attractedto both positively and negatively charged glass surfaces. However, onlynegatively charged glass interacted with the edge of the nanocrystalsindicating the presence of a positive charge on the edge of the NMPnanocrystals. Like clays, the simultaneous presence of positive andnegative charges appears to allow the novel synthesized colloidal NMPdispersion to form a physical hydrogel with a “clay-like” behavior.

Surface Characterization

XPS measurements were carried out with a Thermo K-alpha spectrometer(Thermo Fisher Scientific Inc, East Grinstead, UK) equipped withmonochromatic Al Kα X-rays source operating at 1486.6 eV. Due to thenon-conductive nature of the powder composing the gel, the chargingeffect was minimized using a low-energy flood-gun to provide efficientcharge neutralization. To preclude charging effects in the resultingspectra the binding energy (BE) scale was calibrated from thehydrocarbon contamination using the C1s peak at 285.0 eV. During themeasurement, the residual pressure inside the analysis chamber was1×10⁻⁸ mbar. The survey spectra were recorded with an X-ray beamdiameter size of 400 m and a passing energy of 200 eV, dwell time of 50ms, and energy step size of 1 eV. High resolution spectra were recordedusing a passing energy of 50 eV, dwell time of 50 ms, and energy stepsize of 0.1 eV. The surface depth profile experiment was realized usingan Ar ions gun working at low current and ion energy of 500 eV thatwould produce an etching rate of 0.05 nm s⁻¹ on a surface of TaO₂. Theetching time was 5 seconds and the process repeated for 5 times.Avantage software (5.932v) was used to fit photoelectron spectra using aleast-squares algorithm. The background in narrow range spectra wasaccommodated by a nonlinear Shirley function, and the experimentalcurves were fitted using combinations of Gaussian and Lorentziandistributions (Aguzzi, C. et al., Appl Clay Sci. 2007, 36 (1-3), 22-36.)Quantification was performed on the basis of Scofield's relativesensitivity factors (AlAbbas, F. M. et al., Journal of PipelineEngineering, 2012. 11(1): p. 63).

Biocompatibility of the NMP Colloidal Dispersions

Cell viability of NMP colloidal suspensions was carried out usingprimary human fibroblast (HF) cells. HFs were kindly provided by Dr. C.Doillon (Université Laval). HFs were derived from foreskins afterwritten informed consent which was approved by the Centre HospitalierUniversitaire de Quebec (CHUQ) Ethics Committee. HFs were seeded oncircular coverslips in 24 well-plate (0.8×105 well), cultured in DMEMcell culture media (10% FBS, 1% penicillin-streptomycin) at 37° C. in ahumidified atmosphere of 5% CO₂ and grown overnight. A solution of 10%Alamar-Blue reagent was prepared in cell culture medium DMEM and usedfor the assay. HF cells without materials were used as positive control,NMP without HF cells was used as a blank, and colloidal suspension offormulation A and B were put in direct contact with HF cells for thecell viability test. After 1 day, the cell culture medium was removedand 500 μL of a 10% solution of Alamar-Blue reagent was added to eachwell and incubated at 37° C. in a humidified atmosphere of 5% CO₂ for 3hours. From each sample 100 μL of culture medium was collected anddeposited in a 96 well-plate. The fluorescence intensity was measured at585 nm using a λ_(exc) of 550 nm using a Microplate Reader SpectraMax M2(Molecular Devices, L.L.C. Sunnyvale, Calif., USA). The same procedurewas used for end point day 4 and 7. Live/Dead Assay. HFs were seeded oncircular coverslips in 24 well-plate (0.4×105 well), cultured in DMEMcell culture media (10% FBS, 1% penicillin-streptomycin) at 37° C. in ahumidified atmosphere of 5% CO₂ and grown overnight. The stainingsolution was prepared mixing calcein (2 μmol L⁻¹), Etd-1 (4 mol L⁻¹),and Hoechst 33258 (4 g mL⁻¹). HF cells without materials were used aspositive control, colloidal suspension formulation A and B were put indirect contact with HF cells for the Live-Dead assay. Negative controlHF cells were treated with 70% of methanol for 30 minutes at roomtemperature. The culture medium was removed, and the cells were washedthree times with 500 μL of phosphate buffer solution (PBS). 400 μL ofthe staining solution was added to each coverslip and incubated for 40minutes at room temperature protected from light. Cells were washedagain with PBS (500 μL) and the coverslips mounted on glass slides.Zeiss Axio Imager M2 (Carl Zeiss Microscopy GmbH, Goettingen, Germany)was used to take the photographs of the Live/Dead assay using threedifferent sets of filters; green for calcein, red for Etd-1, and bluefor Hoechst 33258. The same procedure was used for end point day 4 and7. All assays were performed in triplicate.

Genes Expression of Osteoblast Differentiation

Mouse-derived bone marrow cells (mBMCs) were kindly provided by Dr. S.Komarova (McGill University). Animal experiments were performed inaccordance with the McGill University guidelines established by theCanadian Council on Animal Care. Mouse-derived bone marrow cells (mBCMs)were collected from mouse tibia and femora using a procedure previouslydescribed (C57BL6/J, male, 6 weeks old, purchased from CharlesRiver)—see Hussein et al., Bone 2011, 48, 202, incorporated herein byreference. mBMCs were cultured using a procedure previouslydescribed—see Tamimi et al., Acta Biomater. 2011, 7, 2678, incorporatedherein by reference. Briefly, mBMCs were cultured in 75 cm² tissueculture flasks (2.5×10⁶ cells cm⁻²) in MEM (Wisent Inc., Canada) with10% serum (Fisher Scientific, Canada), 1% penicillin/streptomycinantibiotics (Wisent Inc., Canada), 1% sodium pyruvate (Wisent Inc.) and50 g mL-1L-ascorbic acid (Sigma-Aldrich Co., USA). After 7-10 days,cells were detached with trypsin/EDTA (Wisent Inc.) and plated at adensity of 10⁴ cells cm-2 directly onto the surface of the materials orthe tissue culture treated polystyrene (Corning Life Sciences, Lowell,Mass., USA). mBMCs were cultured for 3, 5, 7, 14 and 21 days using MEM10% serum, 1% antibiotics, 1% sodium pyruvate, 50 μg mL⁻¹ ascorbic acid,10 mM glycerol 2-phosphate disodium salt hydrate, and dexamethasone1×10⁻⁹ M. Cell cultures were supplemented with fresh medium every secondday. Total RNA was isolated from mBMC primary cultures using TRI-zol©reagent (Invitrogen™, USA) following the manufacturer's protocol andquantified in a spectrophotometer by absorbance readings at 260 nm. Forreal-time PCR, 1 g of total RNA from each sample was reverse transcribedusing a high-capacity cDNA reverse transcription kit (AppliedBiosystems, USA) in accordance with the manufacturer's instructions. Theresulting cDNAs were used for real-time PCR using Power SYBR Green PCRMaster Mix (Applied Biosystems). Reactions were carried out in a 7500Real-time PCR System (Applied Biosystems) for 40 cycles (95° C. for 15s, 60° C. for 30 s and 72° C. for 45 s) after the initial 10-minuteincubation at 95° C. A cycle threshold value for each reaction wascalculated using Applied Biosystems sequence detections software and therelative ratio of expression was determined using a previously describedalgorithm—see M. W. Pfaffl, Nucleic Acids Res. 2001, 29, incorporatedherein by reference. Primers used to amplify specific targets are asfollows: RunX2 (runt-related transcription factor 2: sense,50-GGCTTGGGTTTCAGGTTAG-30 (SEQ ID NO: 1); antisense,50-CGGTTTCTTAGGGTCTTGGA-30 (SEQ ID NO: 2)), TNALP (tissue nonspecificalkaline phosphatase: sense, 50-GGGGACATGCAGTATGAGTT-30 (SEQ ID NO: 3);antisense, 50-GGCCTGGTAGTTGTTGTGAG-30 (SEQ ID NO: 4)), COL1A1 (collagentype I, alpha 1: sense, 50-GAGGCATAAAGGGTCATCGTGG-30 (SEQ ID NO: 5);antisense, 50-CATTAGGCGCAGGAAGGTCAGC-30 (SEQ ID NO: 6)), OCN(osteocalcin: sense, 50-TGAACAGACTCCGGCG-30 (SEQ ID NO: 7); antisense,50-GATACCGTAGATGCGTTTG-30 (SEQ ID NO: 8)), and OPN (osteopontin: sense,50-CTGCTAGTACACAAGCAGACA-30 (SEQ ID NO: 9); antisense,50-CATGAGAAATTCGGAATTTCAG-30 (SEQ ID NO: 10)).

In Vivo Study of Bone Healing and Implant Osseointegration

The in vivo part of this study was approved by McGill Ethics BoardCommittee in accordance to Canadian Council on Animal. This part wasconducted on thirty-nine 10-12 week-old Sprague-Dawley rats (CharlesRiver Laboratories, Montreal, QC). The rats were housed in Genome Animalfacility of McGill University and caged in a controlled environment at22° C. with 12-hour light/dark cycles and humidity of 50%. A rodentbreeding diet and water were provided ad libitum. All rats were allowedto acclimatize to this environment for 2 weeks prior to surgicalintervention.

Twenty-four animals were used to assess the effect of NMP gel on bonehealing and implant osseointegration. These animals were divided intotwo groups; first control (12 rats) and; second NMP gel (12 rats). Toprovide sufficient analgesia during surgical procedure, rats wereinjected with Carprofen (5-10 mg/kg, subcutaneous, Pfizer Animal Health,Montreal, QC) thirty minutes prior to surgical intervention. The ratswere anesthetized with isoflurane (4% during the induction and 2.5%during the surgical procedure); the legs were shaved, disinfected withchlorhexidine gluconate solution (Omega Laboratories, Montreal, Canada)and covered with a sterile drape. A full thickness incision was made toexpose the proximal third of the tibia. A uni-cortical defect (2.5 mm ø)was created in the right tibia using straight hand-piece under constantsaline irrigation. The same procedure was performed in the contralateralside but custom made titanium (Ti) implant (1.5 mm ø×2.0 mm in depth)was placed in the defect. In NMP group, the Ti implants were coated withNMP gel before insertion in the defect and the contralateral defectswere filled with the NMP gel (20 μL). The implant and the defects werenot treated in the control group. Incisions were sutured using 5-0monocryl sutures. In order to provide sufficient analgesia to the ratsfollowing surgery, they were administered with Carprofen every 24 hoursfor the first 3 days. Rats were allowed to heal for two weeks and thenwere euthanized using CO₂ asphyxiation, and the tibiae were extractedand preserved in 10% neutral buffered formalin (Richard AllanScientific, Kalamazoo, Mich.). Samples were code labeled and codes wereblinded to the person who did the analyses.

Micro-CT

Since the introduction of metallic implants for medical use by Branamarkin 1981 (ref), histology has beenc considered the most accurate methodfor assessing osseointegration. However, histology has severallimitations: it only allows two-dimensional assessment, it is expensive,multi-step processing may be expensive, and it destroys the sample.Therefore, μ-ct was introduced as a method for bone volumetric analysis.However, it had disadvantages too since assessment of the bonesurrounding implants was not possible due to difference in densitiesbetween the bone and the metallic implant. Here, we introduce a newmethod to assess osseointegration using μ-ct. The right tibiae (bonedefect samples) were scanned using a micro-CT (SkyScan1172; SkyScan;Kontich, Belgium) set at 12.7 μm resolution, 50 kV voltage, 200 μAcurrent, 0.5 degree rotation step and 0.5 mm aluminum filter. Theoriginal bone defect (2.5 mm 0, full thickness of cortex) was identifiedas a region of interest (RO). The ROI was analyzed and the volume of thedefect was calculated by subtracting the bone volume from the totalvolume of the ROL.

The left tibiae samples with Ti implants were also scanned using amicro-CT (SkyScan1172; SkyScan; Kontich, Belgium) but set at 4.5 μmresolution, 100 kV voltage, 100 ρA current, 0.4 degree rotation step andaluminum/copper filter. The reconstructed images were segmented bydifferent thresholding to obtain two ROIs. The first ROI included thetitanium implant only by thresholding the intensity of the white colorat low 80 and high 255. The second ROI included the implant/bone in theperi-implant area by an exact dilation of the the first ROI and followedby thresholding the intensity of the white color at low 10 and high 255.The final ROI (the peri-implant bone) was determined by subtracting thefirst ROI from the second ROI and the bone peri-implant area wasanalyzed.

Gene Expression of in vivo Study

Twelve rats were used to assess the effects NMP gel on RunX2 and COL1A1gene expression. Two uni-cortical defects (2.5 mm ø) were created inboth tibiae. The right tibial defect was administered with NMP gel whilethe contralateral side was left empty. COL1A1

Primers used to amplify specific targets are as follows: RunX2(runt-related transcription factor 2: sense, 50-GGCTTGGGTTTCAGGTTAG-30(SEQ ID NO: 1); antisense, 50-CGGTTTCTTAGGGTCTTGGA-30 (SEQ ID NO: 2)),TNALP (tissue nonspecific alkaline phosphatase: sense,50-GGGGACATGCAGTATGAGTT-30 (SEQ ID NO: 3); antisense,50-GGCCTGGTAGTTGTTGTGAG-30 (SEQ ID NO: 4)), COL1A1 (collagen type I,alpha 1: sense, 50-GAGGCATAAAGGGTCATCGTGG-30 (SEQ ID NO: 5); antisense,50-CATTAGGCGCAGGAAGGTCAGC-30 (SEQ ID NO: 6)), OCN (osteocalcin: sense,50-TGAACAGACTCCGGCG-30 (SEQ ID NO: 7); antisense,50-GATACCGTAGATGCGTTTG-30 (SEQ ID NO: 8)), and OPN (osteopontin: sense,50-CTGCTAGTACACAAGCAGACA-30 (SEQ ID NO: 9); antisense,50-CATGAGAAATTCGGAATTTCAG-30 (SEQ ID NO: 10)).

Bone Healing and Osseointegration

Samples with bone defects were dehydrated in ascending concentrations ofethanol (70-95%). Three sections obtained and stained with eithertartrate resistance acid phosphatase (TRAP) to assess the number ofosteoclasts, alkaline phosphatase (ALP) to assess the effects on theosteoblast number, and Von Kossa to assess the mineralization.Histological sections were recorded using an optical microscope (CarlZeiss Microscopy, Germany). The number of osteoclasts and osteoclastswere quantified using an imaging software (ZEN 2012 SP2, Germany).Osteoclasts data were presented as osteoclast number per squaremillimeter of mineralized tissue (OC/mm²). Similarly, the osteoblastsdata were presented as osteoblasts number per square millimeter ofmineralized tissue (OB/mm²). The percentage of mineralized tissue in thedefect was analyzed using Image J (Wayne Rasband; National Institute ofHealth, Bethesda, Md.) and data were presented as mineralized tissuepresent (MT %). All data are presented as mean±standard deviation. Lefttibiae samples with Ti implants were dehydrated in ascendingconcentrations of ethanol (70%-100%) and infiltrated withpoly(methyl-methacrylate) histological resin (Technovit 9100, HeraeusKulzer, Wehrheim, Germany). After polymerization, samples were sectionedinto 30 μm thick histological slides using a diamond saw (SP1600, LeicaMicrosystems GmbH, Wetzlar, Germany) and stained using basicfuchsine andmethylene blue. Histological sectionswere imaged using an opticalmicro-scope (Carl Zeiss Microscopy, Germany) and analyzed using ImageJsoftware (Wayne Rasband; National Institute of Health, Bethesda, Md.).The bone-implant contact was calculated by dividing the bone-coveredimplant perimeter by the total implant perimeter.

Statistical Analysis

The sample size for the in vivo part of the study was calculated to givea power of 80% at 95% confidence level to reject the null hypothesisthat there is no difference between NMP gel-coated and non-coatedimplants. A 10% difference was considered to be clinically relevant, 12%potential standard deviation was assumed based on previous study, and a10% potential drop-out was expected (based on our previous experimentusing similar model and intervention). Shapiro-Wilk test was used toassess data normality and Student's t test was used to compare betweengroups. Origin 9 software (Origin Lab Co., Northampton, Mass.) was usedfor data analyses. Statistical significance was set at p<0.05.

Results

Identification, Characterization, nanocrystals morphology and structureof the NMP phase

The novel 2D Nanocrystalline Magnesium Phosphate (NMP) biomaterial isidentified in the ternary diagram of the system NaOH—Mg(OH)₂—H₃PO₄ (FIG.2).

NMP gels were obtained in a small region of the ternary diagram (FIG. 3,area labelled “Stable colloidal suspension”). The symbols on this areaof the ternary diagram refer to specific formulations A=

, B=

, C=

, and D=

reported in Table 2.

Depending on the mole fractions used, the remaining crystals phasesidentified ranged from di- and tribasic magnesium phosphate such asNewberyite MgHPO₄.3H₂O, Farringtonite Mg₃(PO₄)₂, BobierriteMg₃(PO₄)₂.8H₂O, and Brucite Mg(OH)₂. In addition, a new unidentifiedcrystal phase (X-ray diffraction pattern shown in FIG. 4) and unstablegel-like colloidal suspension were obtained (FIG. 3, are a labelled “Newcrystal phase and mixed Mg/PO₄ phases” surrounding the area labelled“Stable colloidal suspension”).

Solutions with a mole fraction (η) of H₃PO₄ higher than 0.8 were tooacidic and no precipitates were obtained (see FIG. 3, black area on theright hand side of the diagram).

The composition of the NMP was comprised of a range of [Mg⁺], [Na⁺],[PO₄ ³⁻], and [HPO₄ ²⁻] and the formula can be assumed to beMg_(X)Na_(Y)(HPO₄ ²⁻)_(Z).(PO₄ ³⁻)_(T).nH₂O with [5≤X≤7], [1≤Y≤2],[3≤Z≤5], and [1≤T≤3] where [7≤X+Y≤8] and [5≤Z+T≤6].

The crystallization water was determined by thermogravimetric analysisand was comprised between [3≤n≤4] (Table 2 and FIG. 5).

TABLE 2 Composition of the four different formulations obtained asstable colloidal suspension measured using ICP-OES. The amount of waterof the dried powder was determined using TGA. The symbols refer tospecific formulations showed in the area labelled ″Stable colloidalsuspension″ in FIG. 3. Mole fraction Atomic Atomic Atomic FormulationH₃PO₄/NaOH/Mg(OH)₂ % P % Na % Mg pH A  

0.35/0.52/0.13 16.2 ± 1.3 6.5 ± 0.2 17.5 ± 0.4 8.3 ± 0.1 B  

0.37/0.45/0.18 19.5 ± 2.3 3.3 ± 0.4 19.5 ± 2.2 7.8 ± 0.1 C   

 0.3/0.55/0.15 20.3 ± 3.0 3.4 ± 1.0 24.7 ± 3.5 10.9 ± 0.1  D  

 0.3/0.52/0.18 20.4 ± 1.5 3.2 ± 1.5 22.2 ± 1.8 10.8 ± 0.1 

The atomic ratio Mg/P was between 1 and 1.15, suggesting that the NMPgel might present similarity with the crystalline precipitate NewberyiteMgHPO₄.3H₂O.

The evolution of various physical aspect of the NMP colloidal suspensionduring the synthesis was as follows. FIG. 6 shows the pH of thecolloidal suspension as a function of the reaction time. The molefraction of H₃PO₄NaOH/Mg(OH)₂ was 0.37/0.45/0.18 and the total volumewas 7 mL. FIG. 7 is a picture of the suspension after 30 seconds fromthe beginning of the reaction. The colloidal suspension was white andliquid, visible by tilting the tube. FIG. 8 shows that after 10 minutes,the NMP colloidal suspension transformed from liquid to solid changingits color from white to gray. Finally, FIGS. 9 A and B show the NMPnanocrystals evolution during the reaction. The SEM micrographs show theevolution of the morphology nanocrystals after 30 s (d) and 30 minutes(e). The scale bar represents 500 nm.

The zeta potential of the NMP nanocrystals showed an overall negativecharge comprised between −10.1±4.3 and −18.1±6.7 mV.

Rheological measurements confirmed the thixotropy of the NMP colloidalsuspension with a solid content of 5-10 wt. % (FIGS. 10 to 14). Afterliquefaction (G′≤G″), t_(L)-G is the time taken for G′, G″ and 0 toreturn to their original levels when shear stress is removed.

At low stress the material remained in solid state, as indicated byG′>G″ (δ<45°), and upon increasing stress it started to liquefy (G′=G″and θ=45°). The minimum shear stress required for this to occur isdefined as the liquefaction stress τ_(y) and its value ranged between 22and 127 Pa. The increase of τ_(y) and viscosity is usually attributed tothe increasing number and extent of interactions between thenanocrystals due to their growing association. After stress removal, thematerial recovered the gel-like state with a recovery time (t_(L-G)) asquick as 12 s which is faster than water dispersion of 2D clay Laponite(20 minutes). T varied according to the viscosity because Brownianmotions are inversely proportional to the medium viscosity; higherviscosity decreased the mobility of the nanocrystals increasing thet_(L-G) needed to reform the original 3D network.

In addition, viscosity was inversely proportional to the size of thenanoparticles, so by varying the mole fraction of the three componentsit was possible to modify the rheological and physical properties of theNMP suspensions (Table 3).

TABLE 3 Physical properties of NMP hydrogels synthesized with differentmole fractions. Zeta Storage Mole fraction Crystal Size PotentialModulus Liquefaction Formulation H₃PO₄/Mg(OH)₂ L × W (nm) (mV) G′ (Pa)Stress Ty (Pa) t_(L-G) A 2.7 285 ± 133 × 54 ± 18 −10.7 ± 0.5 1769 22 12s B 2.1 237 ± 83 × 64 ± 25 −10.1 ± 4.3 4316 56 60 s C 2 156 ± 84 × 39 ±8 −17.4 ± 5.5 5199 80 95 s D 1.6 141 ± 82 × 39 ± 12 −18.1 ± 6.7 10540127 >300 s

The NMP gel could be injected easily through an insulin needle and aftermanual injection the colloidal suspension would regain solid-likebehavior (FIG. 15). The force required to inject the NMP materialthrough the insulin needle was only 0.08-0.63 N more than the forcerequired to inject water (0.14±0.03 N Experimental Section, above).After flipping the glass slide, the suspension behaved like a solidmaterial.

The real 3D structure of the colloidal suspension was revealed with TEMfreeze-etching fracture technique due to the inherent sample techniquepreparation. The nanocrystals are kept in place by the underlying icematrix, and after being exposed for replication, the carbon-metalreplica shows the network formed by the nanocrystals. Low magnificationTEM micrograph of the carbon-metal replica of a freeze-fracturedsuspension shows bundled NMP nanocrystals forming the 3D network, thewater composing the medium of the hydrogel being in the empty spacebetween the bundled nanocrystals (FIG. 16). The surface of the replicashowed a stepped pattern revealing the thickness of the nanocrystalsthat was estimated to range between 4 and 7 nm (FIG. 17).

TEM micrographs illustrated the high aspect ratio of the nanocrystals,and selected area electron diffraction showed the nanocrystalline natureof the biomaterial (FIGS. 18 to 22).

One of the models used to explain thixotropy is the aforementioned“house of cards”, where the particles are held together by edge-to-facecontacts due to the simultaneous presence of positive and negativecharges on the edge and face of the particles. High magnification TEMmicrograph reveals that the type of aggregation of the nanocrystalsmainly consist of face-to-face associations. The nanocrystals partiallyoverlap each other resulting in a honeycomb network of an extendedsheet-like face-to-face aggregates that are bent, twisted, branched, andintertangled with few edge-to-face contacts (FIGS. 17 and 23).

The very thin structure of the 2D nano-sheet is visible on the TitanKrios micrograph of the NMP gel (FIG. 24).

Sodium Ions Stabilize NMP Nanocrystals

The synthesis of the thixotropic NMP biomaterial was achieved usingdifferent alkaline bases (NaOH, KOH, and LiOH) and the material showedvery long term stability (>1500 days, 20° C.). However, the stabilitywas dramatically influenced by the type of alkali ion used. Smaller ionssuch as Li⁺ and Na⁺ (ionic radius 76 and 102 μm, respectively) producedstable hydrogels, whereas larger K⁺ ions (ionic radius 138 μm) resultedin unstable hydrogels that eventually converted to NewberyiteMgHPO₄.3H₂O (FIG. 28). The replacement of Na⁺ with K⁺ ions affected thestability of the suspension that decreased from >4 years to <1 dayresulting in a quick conversion of NMP to Newberyite (FIG. 25); thespeed of conversion was directly correlated to the ratio[Na]/([K]+[Na]). The stable NMP suspension made with sodium remained inthe gel state and it had no phase change after 4 years as shown by XRD(FIG. 26). Both NMP and Newberyite have similar Mg/P ratio, they form ina similar pH range 6.4-9.4 (FIG. 27, arrows).

NMP contains both HPO₄ ²⁻ and P₄ ³⁻. This was demonstrated by performingonto the washed NMP powder FT-IR, solid state magic angle spinning (MAS)NMR, and XPS.

The FT-IR spectrum of formulation A (FIG. 29) showed a broad absorptionsband from 3600 to 2600 cm⁻¹, indicating a combination of 0-H stretchingof intermolecular and weakly bonded crystallization water and stretchingof (P)O—H. As in other hydrogen phosphates (e.g. Monetite CaHPO₄) the OHstretching of the HPO₄ ²⁻ anion give rise to at least two broad bandsaround 2855 and 2385 cm⁻¹. The band around 2855 cm⁻¹ was covered by thebroad absorption of the water molecules, while the band at 2355 cm⁻¹ wasvisible. The broad absorption at 1647 cm⁻¹ was also attributed to acombination of HOH bending and rotation of residual crystallization andfree water. The absorptions bands at 1080 and 1005 cm⁻¹ were associatedwith the stretching vibrations of P═O, the band at 875 cm⁻¹ wasattributed to the bending of P—O—H, while the band at 535 cm⁻¹ wasattributed to the bending of P═O.

FIG. 30 shows the FT-IR spectrum of the same powder after calcination at700° C. for 8 hours. As can be observed, the vibrations of the P—O—Hmoiety disappeared and new additional vibrational modes appeared in thefrequency range of 1250-450 cm⁻¹ that were assigned to the presence ofP₂O₇ ⁴⁻, demonstrating the transformation of HPO₄ ²⁻ to P₂O₇ ⁴⁻.

The ³¹P spectra (FIG. 31 top) revealed two peaks. The left one withoutvisible sidebands was assigned as P₄ ³⁻, whereas the right one withsidebands presented a stronger signal at the CP experiment and it wasassigned as HPO₄ ²⁻. ²³Na spectra taken with two different recycledelays (50 ms and 1 s) are shown in FIG. 31 bottom. One of the peaks wasmore evident at RD=50 ms. MAS NMR showed that, HPO₄ ²⁻ cross polarizesintensely due to the small distance between hydrogen and phosphate ionsin its chemical molecular structure. Therefore, HPO₄ ²⁻ yields astronger signal in the CP MAS spectra, as compared with PO₄ ³⁻. On theother hand, PO₄ ³⁻ yields a stronger and narrower signal in the regular³¹P MAS spectra with no detectable sidebands, indicating the isotropicnature of the crystals of this ion and the presence of a smallerspin-spin coupling effect.

After immersing the NMP gel in the D₂O signal from HPO₄ ²⁻ has beenprogressively weakened, with no remaining signal found in the experimentwith 14 hours in D₂O, which indicates the protons exchange between thismolecule and D₂O. On the other hand, all signals from PO₄ ³⁻ have beenbroadened, and now presenting short and broad sidebands (more clearlynoticeable in the CP experiments). This may have occurred due to thealteration of the chemical environment due to the presence of D₂O,causing different isotropic chemical shifts for PO₄ ³⁻ and leading to aless organized crystallographic structure (FIG. 32).

High resolution XPS spectra P2p confirmed the presence of two differentkinds of phosphate anions, and its deconvolution into two peaks at 133.7and 134.9 eV were assigned respectively to PO₄ ³⁻ and HPO₄ ²⁻ (FIGS. 33(a) and (b)).

XPS depth profile (FIG. 33 (c)) also revealed that the concentration ofsodium slightly decreased from 9.1 to 7.6 at. % after mild etching usingAr ions at very mild condition. The superficial excess of sodium on theouter surface of the nanocrystals could be due to the presence ofnegative charges on the faces of the nanocrystals, and those ions mightbe responsible of the stabilization of the metastable nanocrystallinephase.

The deposition of NMP powder onto different treated glass surfaces wasalso studied. FIG. 34 shows the deposition of NMP on a negativelycharged glass surface. The nanocrystals can adopt a parallel orperpendicular direction to the surface. FIG. 35 show NMP powderdeposited on a positively charged glass surface. The nanocrystals adoptonly a parallel configuration to the glass surface. In addition, theamount of nanocrystals deposited onto the substrate surface withnegative charge was considerably higher than the positive surface. Notethe contrast in FIGS. 34 and 35 was enhanced for visibility in black andwhite.

Cytocompatibility of NMP Colloidal Suspension

We further investigated the cytotoxicity of the NMP nanocrystals bymeans of live-dead assay and Alamar-Blue assay to measure the metabolicactivity. Alamar-Blue assay on Human Fibroblast cells seeded in directcontact with the NMP gel revealed that the metabolic activity offormulation A and B increased from day one to day seven (FIG. 36).Formulation B showed a higher metabolic activity than formulation Aduring the seven days of the experiment, and at day seven no significantdifference was observed between the control and formulation B. Thehigher value of formulation B might arise from its more physiological pH(7.8) than formulation A (8.3) Live-dead assay results suggested goodcell viability for the tested formulations A and B (FIG. 37 to 41).

Note that the order of the bars in FIGS. 36 and 37 from left to right isthe order shown in the legend in FIG. 36 from top to bottom. In FIGS. 38to 41, contrast was enhanced for visibility in black and white.

SEM micrograph shows the morphology, adhesion, and colonization ofdifferentiated osteoblasts from mouse bone marrow cells (mBMCs) culturedonto NMP biomaterial. After eight days of culture, the image showscell-cell and cell-substrate interactions that enabled the formation ofa macro-scale tissue construct (FIG. 42). Moreover, comparing thestructure of the NMP nanocrystals before and after cells culture, it canbe observed an increase of the nanocrystals porosity probably due to thedissolution of the NMP nanocrystals.

The NMP gel synthesized in formulation B (pH 7.8) was further studiedfor bone formation purposes. Runt related transcription factor 2 (RunX2)is considered a key factor of osteogenesis due to the stimulation ofosteoblast-related genes such as alkaline phosphatase (ALP), osteocalcin(OCN), osteopontin (OPN), and collagen, type I, alpha1 (COL1A1). Theregulation activity of ALP is a key event that occurs during the earlytime of osteogenesis. Our result shows an early increase andup-regulation of ALP during the first five days respect MgHPO₄.3H₂O andMg₃(PO₄)₂.22H₂O indicating that NMP had a positive effect on ALPexpression and promoted osteogenic differentiation (FIG. 43).

The synthesis of OCN and OPN is also a key indicator for bonemineralization. OCN is secreted solely by osteoblasts and regulates bodymetabolism and bone building process, being the most specific marker forosteoblast differentiation and mineralization. Real Time-PCR of OCNshowed that during the first 14 days, mBMCs cultured on NMP expressedsignificant higher levels of the gene than cells cultured on MgHPO₄.3H₂Oand Mg₃(PO₄)₂.22H₂O (FIG. 44).

OPN or bone sialoprotein is a structural protein that accounts ˜8% ofall non-collagenous proteins found in bone, and it is mainly synthesizedby pre-osteoblasts, osteoblasts and osteocytes. Our results shown thatNMP up-regulated the expression of OPN up to 21 days (FIG. 45).

We also followed the mRNA expression of COL1A1, a gene responsible toencode the production of pro-alpha1(I) chain of type I collagen that isa constituent of the ECM in connective tissue such as bone, skin,tendon, ligament and dentine. NMP up-regulated COL1A1 expressed by mBMCsreaching a maximum at day 7 in comparison to MgHPO₄.3H₂O andMg₃(PO₄)₂.22H₂O (FIG. 46). mBMCs cultured on NMP expressed higher levelsof RunX2 than mBMCs cultured on Cattiite (Mg₃(PO₄)₂.22H₂O) andNewberyite after five days of incubation (FIG. 47).

In FIGS. 43 to 47, differences were assessed by one-way analysis ofvariance and accepted as statistically significant at p<0.05*.

The novel NMP material has osteogenic properties and can trigger aseries of events such as osteoblast cell proliferation, collagensynthesis, ECM maturation, and mineralization which follow the temporalpattern of osteogenic differentiation. The 2D material up-regulates themRNA expression of RunX2/ALP and also the genes responsible of theformation of the extracellular matrix with bone-related protein OCN,OPN, and type I collagen.

NPM Accelerates Bone Healing and Implant Osseointegration

NMP effects on bone formation (healing and osseointegration) were alsoinvestigated in vivo using rats' tibiae model. Computerizedmicro-tomography (μ-CT) scans were performed on bone samples that wereretrieved at different time points 3, 7 and 14 days after surgery forNMP treated and non-treated defects. At day 3, there is no visibledifference on μ-CT scan between the control and NMP group. On the otherhand, at day 7 the μ-CT scan clearly indicates that the tibial defecttreated with NMP shown more bone formation in the defect compared to thecontrol ones (FIG. 48). After 14 days, the tibial defect treated withNMP is almost fully filled with new bone while the control is stillpartially healed.

Histology and histomorphometry analysis showed that NMP accelerated bonehealing and enhanced osseointegration (FIGS. 49 to 53 and Table 4)through up-regulation of osteoclasts proliferation (FIGS. 49 and 53),osteoblasts differentiation (FIGS. 49 and 54), collagen synthesis (FIGS.49 and 51) and mineralization (FIGS. 48 and 49). μ-CT and histology ofimplant show more bone in contact with implant (FIGS. 54 and 50).Histology and histomorphometry indicate that NMP clearly enhanced thebone forming cells; osteoblast and osteoclasts as well as collagen.

TABLE 4 μ-CT analyses show smaller defect volume, less trabecularseparation (Tb.Sp), more trabecular thickness (Tb.Th) and trabecularnumber (Tb.N) in NMP-treated defects. Group NMP Control Defect Vol.(mm³) 1.51 ± 0.20 2.19 ± 0.32 Tb.Th (mm) 0.30 ± 0.04 0.25 ± 0.04 Tb.Sp(mm) 0.11 ± 0.02 0.15 ± 0.05 Tb.N (mm⁻¹) 2.52 ± 0.25 1.98 ± 0.61

Focus Ion Beam SEM (FIB-SEM) was performed on defect samples that wereretrieved at day 3 and 7. At day 3, no mineralization was present inboth group, however at day 7, FIB-SEM images show collagen bone matrixundergoing mineralization by osteoblasts in NMP-treated defect (FIGS. 55and 56).

NMP effects on genes expression (COL1A1 and RunX2) were further assessedin vivo by treating rats tibial defect with NMP. Bone samples wereretrieved and assessed by qRT-PCR at different time points; 3, 7 and 14days and showed that expression of COL1A1 and RunX2 were significantlyup-regulated already at day 3 following surgery (FIGS. 57 and 58).

Osteoblast differentiation was up-regulated (FIG. 52) due to the NMPeffects on osteoblastic gene markers ALP, OPN and RunX2 (FIGS. 43, 45,47, and 57). The presence of magnesium ions increases the expression ofosteoblast phenotype genes. Magnesium increases the expression of ALP,OPN and RunX2. On the other hand, calcium increases the expression ofColl.

Osteoclasts proliferation was also enhanced by NMP (FIGS. 49 and 53).NMP, which is rich of magnesium, enhanced osteoclasts proliferation andmineralization of the healing defects.

Collagen synthesis was also up-regulated in NMP treated defects (FIGS.49 and 51) because NMP enhanced expression of COL1A1 (FIGS. 46 and 58).

Conclusions

In summary, we synthesized a nanocrystalline 2D biomaterial made ofMg—Na—(HPO₄)—(PO₄). Water dispersions of NMP nanocrystals results in theconstitution of a physical hydrogel that had thixotropic behavior andwas easy to inject.

The physical properties of this novel material can be tailored byvarying the mole fraction of NaOH—Mg(OH)₂—H₃PO₄.

Cell viability assays with human fibroblasts cells in direct contactwith the NMP hydrogel showed its biocompatibility. In vitro mRNAexpression of mBMCs cultured on NMP showed the up-regulation of genes(RunX2, ALP, OPN, OCN, and COL1A1). Moreover, it enhanced bone healingand osseointegration by stimulating differentiation and activity of bonecells; osteoblast, osteoclasts and collagen.

Example 2—Synthesis of Further Gels

Here, we describe the synthesis of nanocrystalline materials withplatelet morphology. The 2D nanocrystals were characterized using avariety of analytical methods. The nanomaterials are based on a novelfamily of MIM(HPO₄)²⁻(PO₄)³⁻ where M^(I)=Na⁺ and M^(II)=Mg²⁺ alone orcombined with Fe²⁺.

The synthesized nanocrystals formed colloidal suspensions that behavedas physical hydrogels presenting long term stability, thixotropy,injectability, and high surface area. Here, we further show an easy andscalable method to synthesize 2D nanomaterials obtained by a simpleprecipitation route. The easiness of synthesis and the interestingphysicochemical properties of these materials open promising applicationin catalysis, layer by layer deposition, and electrodes for batteries.

Experimental Section Materials and Methods

We synthesized different M^(I)M^(II)(HPO₄)²⁻(PO₄)³⁻ where M^(I)=Na⁺ andM^(II)=Mg²⁺ alone or combined with Fe²⁺.

FeCl₂.4H₂O and Mg(OH)₂ were purchased from Sigma-Aldrich (Milwaukee,Wis., USA).

2D Nanocrystals of MgNa(HPO₄)²⁻(PO₄)³⁻

Stable colloidal suspension of 2D nanocrystals was done using thefollowing conditions. 85 mg of Mg(OH)₂ (1.45 mmol, mole fraction 0.14)were dissolved in 2.2 mL of H₃PO₄ 1.5 M (3.30 mmol, mole fraction 0.31).After complete dissolution of Mg(OH)₂, 3.8 mL of NaOH 1.5 M (5.7 mmol,mole fraction 0.55) were added under vigorous stirring. The addition ofNaOH provoked the instantaneous formation of a white colloidalsuspension that after 6 minutes changed to a solid state with a greycolor. The final pH of the colloidal suspension was 8.46. After 2 hours,the colloidal suspension was centrifuged at 4000 rpm for 5 minutes andthe supernatant was discarded. The solid precipitate was washed withethanol to remove the excess of water, vacuum dried at room temperature,and stored for characterization.

Combination of FeCl₂.4H₂O and Mg(OH)₂

60 mg FeCl₂.4H₂O (0.3 mmol, mole fraction 0.02) and 90 mg of Mg(OH)₂(1.54 mmol, mole fraction 0.12) were dissolved in 2.6 mL of H₃PO₄ 1.5 M(3.9 mmol, mole fraction 0.31) and to this solution were added 4.5 mL ofNaOH 1.5 M (6.75 mmol, mole fraction 0.55) to yield a pale greencolloidal suspension. The final pH was 8.10. The mixture was processedas described for Mg.

Synthesis of MgNa(HPO₄)²⁻(PO₄)³⁻ Colloidal Dispersion Using DifferentSources of Mg

The colloidal dispersion with Mg was also obtained by using magnesiumchloride (MgCl₂.6H₂O) instead of magnesium hydroxide (Mg(OH)₂). Thereaction was carried out by dissolving 667.5 mg of MgCl₂.6H₂O (3.275mmol) in 2.5 mL of deionized water and 935 mg of Na₂HPO₄ (6.525 mmol) in10 mL of deionized water. After dissolving the solids, the Na₂HPO₄ waspoured into the solution of MgCl₂.6H₂O under stirring. The resultingcolloidal dispersion had a white color and after 20 minutes from thebeginning of the reaction a grey thixotropic gel was obtained.

NaMg(HPO₄)²⁻(PO₄)³⁻ was also obtained by replacing Mg(OH)₂ withmagnesium oxide (MgO). Briefly, 160 mg of MgO were dissolved in 11.2 mLof H₃PO₄ 1.5 M and 16.8 mL of NaOH 1.5 M were added under vigorousstirring. The colloidal suspension had a white color and after 10minutes a grey thixotropic gel was obtained.

The replacement of NaOH with sodium tripolyphosphate (Na₅P₃O₁₀) alsoallowed the production of the M^(I)M^(II)(HPO₄)²⁻(PO₄)³⁻ gel. Briefly,110 mg of MgCl₂.6H₂O (0.545 mmol) were dissolved in 2.5 mL of deionizedwater and 12.5 mL solution of NaOH 0.2 M and Na₅P₃O₁₀ 15% were addedunder stirring.

Large Scale Mixtures

Most of the previously described colloidal suspension can be made insmall volume batches of 50 mL. Using the conditions reported, we studiedthe scalability of the synthesis using a simple “2-in-” system with onetube connected to a reservoir of a NaOH solution (1.5 M) and the othertube connected to a reservoir of H₃PO₄ (1.5 M) solution containingMg(OH)₂ (0.6 M). To obtain the NaMg(HPO₄)²⁻(PO₄)³⁻ colloidal suspension,the minimum flow velocity of the two solutions inside the tube toprovoke a turbulent regime with a Reynolds number was >4000. Under theseconditions, the turbulent flow at the intersection point provides a goodmix of the two solutions and a method for the continuous production ofthe colloidal suspension.

Characterization

X-ray diffraction patterns of the dried precipitates were recorded witha Bruker D8 Advance (Bruker AXS GmbH, Karlsruhe, Germany) from 50 to 58°2θ with a copper source (λ_(Cu,Ka)=1.54 Å) at 40 kV and 40 mA and GADDSdetector. The diffraction patterns of the dried precipitates containingFe were recorded with a Bruker D8 Discover from 50 to 58° 2θ with acobalt source (λ_(Cu,Ka)=1.79 Å) at 35 kV and 45 mA and GADDS detector.The diffraction patterns were processed with EVA software (Bruker AXSGmbH, Karlsruhe, Germany) and phase composition was determined bycomparing the acquired spectra with the phases identified in theInternational Centre for Diffraction Data (ICDD) database PDF-4.

Fourier Transform Infrared Spectroscopy (FT-IR) of the driedprecipitates were recorded using a Perkin Elmer Spectrum Two (PerkinElmer Inc, Waltham, Mass., USA) with single bounce diamond forAttenuated Total Reflectance (ATR). Spectra were recorded at roomtemperature from 450 to 4000 cm⁻¹ with a resolution of 4 cm⁻¹ and 64scans.

Thermogravimetric analysis (TGA) was performed to calculate the amountof crystallization water of the dried precipitates (SDT Q600 TAInstruments, TA Instruments-Waters L.L.C. New Castle, USA). TGA was donein vertical mode on a platinum pan from 30 to 800° C. using a heatingrate of 5° C./min, and in air atmosphere with a purge flow rate of 100mL min⁻¹.

Zeta potential measurements were carried out to assess the superficialcharge of the nanocrystals using a Malvern Nano ZS equipped withdisposable folded capillary cells (Malvern Instruments Ltd, Malvern,UK). The nanocrystals concentration used for the measurements was set to20 mg mL⁻¹ and the temperature was kept constant at 25° C.

The morphology of the different nanocrystals obtained was revealed byScanning Electron Microscopy (SEM) using a FEI Inspect F-50 FE-SEM (FEIInc, Hillsboro, Oreg., USA) operated at 10 kV. Prior analysis thesamples were sputtered achieving a homogenous coating layer of 2 nm Pt(Leica EM ACE600, Leica Microsystems Inc, Concord, Ontario, Canada). Theelemental composition of the colloidal suspension was determined usingenergy dispersive X-ray analysis spectroscopy (EDS) performed with anEDAX Octane Super Silicon Drift Detector and analyzed using TEAM™software version 4.0.2 (AMETEK, Inc. Berwyn, Pa., USA). The device wasinstalled on a F-50 FE-SEM (FEI Inc, Hillsboro, Oreg., USA) operated insecondary electron mode at 10 kV. ZAF (atomic number (Z), absorption(A), and fluorescence (F)) standard-less analysis was carried out oneach of the EDS spectra using eZAF Smart Quant Results Acquisition(TEAM™ software version 4.0.2) to determine the atomic percentage (at.%) of the washed nanocrystals. The EDS spectra were acquired at 10 kVfor −2 min with a count rate of ˜3000 counts/s.

The specific surface area of the products was measured by theBrunauer-Emmett-Teller (BET) method using nitrogen adsorption anddesorption isotherms on an automated gas adsorption analyzer Tristar3000 (Micromeritics Instrument Corporation Norcross, Ga., USA).

The force required to inject the thixotropic colloidal suspensionsthrough an insulin needle of 160 μm of internal diameter was measuredusing a Mach-1 V500cs and Mach-1 Motion software version 4.3.1(Biomomentum Inc., Laval, Canada). The force was measured with amultiple-axis load cell of 70 N (resolution of 0.007 N) and acquisitionrate of 100 Hz. The gel was loaded into the syringe avoiding thepresence of bubbles and then the plunger was inserted into the loadcell. The force value was measured applying a constant vertical stagevelocity of 1 mm s⁻¹ (resolution of 0.1 μm).

Results and Discussions

The colloidal suspensions formed physical gels.

The colloidal dispersions showed a thixotropic behavior forming aphysical gel. The nanocrystals synthesized presented a two-dimensionalmorphology that to form a physical hydrogel with a thixotropic clay-likebehavior. Clays are plate-like poly-ions with a heterogeneous chargedistribution that forms a physical gel in water at concentrations higherthan 40 mg/mL due to the simultaneous presence of positive and negativecharges that give rise to electrostatic and Van der Waals interactions.This allows the gel to behave as a thixotropic material due to theformation of a 3D network of particles known as the “house of cards”structure. Thixotropic materials can be liquified by applying mechanicalenergy allowing the physical gel to behave as a liquid; then when themechanical stress is removed Brownian motions drive the particles intocontact to reform the 3D network and the liquefied dispersion becomesgel-like again. Like clays, the simultaneous presence of positive andnegative charges allowed the novel synthesized nanocrystals dispersionto form a physical hydrogel with a “clay-like” behavior. As a result ofits unique rheological property, the NMP gel could be injected easilythrough an insulin needle and after manual injection the colloidalsuspension would regain solid-like behavior. After injection thematerial rapidly recovers its solid state and behaves again as solid,this is an important feature for coatings applications procedure.

The FT-IR data, X-ray diffraction pattern of the dried and washedpowders demonstrated the nanocrystalline nature of the 2D nanocrystalsdue to the presence of broad diffraction peaks.

Example 3—from Toothpaste to “Implant-Paste”: A New Product for CleaningDental Implants Abstract

This study aimed at developing an organic-free prophylaxis pasteoptimized for cleaning dental implants (hereinafter called the“implant-paste”), while preserving their surface integrity.

The implant-paste was developed by combining an inorganic thickeningagent made of a nanocrystalline colloidal suspension (NanocrystallineMagnesium Phosphate) and polishing nanoparticles of hydrated colloidalsilica. The implant-paste formulation was optimized to decontaminatetitanium surfaces coated with oral biofilm and compared to a commercialtoothpaste (Colgate Total; Colgate-Palmoliven, USA). Surface morphology,bacterial load and attachment and chemical properties of titaniumsurfaces were analyzed and comparisons between different products weredone using one-way ANOVA and independent samples t tests.

An inorganic prophylaxis paste made of nanocrystalline magnesiumphosphate gel (10% w/w) and (30% w/w) hydrated silica was superior tobrushing alone and Colgate toothpaste in removing titanium surfacescontaminants and it did not cause surface alteration. The thixotropicand inorganic nature of the nanocrystalline magnesium phosphateimplant-paste is ideal for cleaning implant surfaces because, unlike theColgate and other commercial toothpastes, it does not containorganic-based thickeners that can adhere tightly on titanium surfacesand thus change their surface chemistry and moreover, does not abradetitanium.

Introduction

Nanocrystalline magnesium phosphate (NMP) gel is a novel inorganiccolloidal suspension. It is stable biocompatible and thixotropic. NMPgel is silicate-free unlike other thixotropic inorganic materials suchas silicate clays that could be more abrasive on implant surfaces. Thisnovel gel is also rich in Na⁺ cations that have toxic effect on bacteriaand can disturb the biofilm structure by displacing the divalent cations(Ca⁺⁺). Conventional toothpastes comprise fluoride that can corrode Ti,organic compounds that can alter its surface chemistry and abrasivesthat can damage its surface microtexture. Accordingly, we hypothesizedthat prophylaxis pastes free of fluoride and organic compounds would bemore efficient for cleaning dental implants. Thus, this study aimed atdeveloping and optimizing a new “implant-paste” specifically designedfor decontamination of dental implant.

Materials and Methods

The study design was reviewed and approved by the Research Ethics BoardCommittee of McGill University (application 14-464 GEN). All subjectsparticipating in this study have signed informed written consents beforetheir participation.

Materials Synthesis

The implant-paste was developed by combining a thickening agent made ofan inorganic nanocrystalline magnesium phosphate (NMP) gel withdifferent concentration of an abrasive agent of hydrated silicananoparticles. In a typical procedure to synthesize the NMP gel, 300 mgof Mg(OH)₂ (5.14 mmol; Mole fraction=0.144 were dissolved in 8.1 mL ofH₃PO₄ 1.5 M (12.15 mmol; Mole fraction=0.34), followed by the additionof 12.3 mL NaOH solution 1.5 M (18.45 mmol; Mole fraction 0.516). Inanother typical procedure, 270 mg of Mg(OH)₂ (4.62 mmol; Molefraction=0.13) was dissolved in 7.5 mL of H₃PO₄ 1.5 M (11.25 mmol; Molefraction=0.31), followed by the addition of 13.5 mL NaOH solution 1.5 M(20.25 mmol; Mole fraction=0.56). The addition of the NaOH solutionprovoked the instantaneous formation of a white liquid suspension madeof nanocrystals with a uniform size of 50 nm. The pH of the suspensionremained constant for 4 minutes (8.3) then slowly decreased andstabilized at 7.8 after 30 minutes for the first procedure, while itremained constant for 4 minutes at 10.1 then slowly decreased andstabilized at 9.6 after 30 minutes for the second procedure. The liquidsuspension changed its color from white to grey and possessed a solidand thixotropic behavior with the final suspension composed of 2Dnanocrystals with an undulate structure. The solid content of the pastewas then modified by adding 20, 30, 50, or 60% of hydrated silicananoparticles with average aggregate particles size of 0.2-0.3 μm. Theaddition of hydrated silica nanoparticles increased the viscosity of thegel depending on the concentration used, however, the thixotropicbehavior and pH of the initial gel were not affected (see FIG. 59A).

Indeed, the photographs of a rotary brush loaded with the NMP gel, thedeveloped implant-paste and Colgate toothpaste (a-c, respectively) andthe photographs of Eppendorf tubes containing the NMP gel, implant-pasteand Colgate toothpaste (d-f, respectively) clearly illustrate that thegel and developed implant-paste are more thixotropic than Colgatetoothpaste. The Colgate toothpaste flew without applying mechanicalshear while the other pastes did not flow.

Samples Preparation

Machined and polished titanium discs (grade 2, Ø 5.0 and 1.0 mm thick;McMaster-Carr, Cleveland, Ohio, United states) were used in this study.The discs were sequentially ultrasonicated in deionized water, acetoneand ethanol for 15 minutes each, before drying over-night in a vacuumoven (Isotemp, Fisher Scientific, USA).

Biofilm Contamination

The biofilm was developed on the titanium surfaces following apreviously described standard protocol—see Gosau, M., et al., Effect ofsix different peri-implantitis disinfection methods on in vivo humanoral biofilm. Clinical Oral Implants Research, 2010. 21(8): p. 866-872;Idlibi, A N., et al., Destruction of oral biofilms formed in situ onmachined titanium (Ti) surfaces by cold atmospheric plasma. Biofouling,2013. 29(4): p. 369-379; Rupf, S., et al., Removing biofilms frommicrostructured titanium ex vivo: a novel approach using atmosphericplasma technology. PloS one, 2011. 6(10): p. e25893; andGrößner-Schreiber, B., et al., Modified implant surfaces show differentbiofilm compositions under in vivo conditions. Clinical oral implantsresearch, 2009. 20(8): p. 817-826, all of which are incorporated hereinby reference.

Alginate impressions were taken to produce study models for eachparticipant's upper jaw. A 1-mm-thick thermoplastic copolyester splintscovering all maxillary teeth were produced. The splints were used to fixTi discs at the buccal aspect of the premolar and molar areas, eachsplint housed 12 Ti discs. The participants were asked to wear thesplints for 24 hours in order to allow for soft biofilm to accumulate onTi surfaces. The participants were instructed to remove and store thesplints during drinking or eating in phosphate buffered saline. After 24hours, the splints were collected, the discs were washed with sterilesaline solution (9%) and stored for further analysis.

Samples Cleaning

A rotary brush was used to clean biofilm-contaminated samples withwater-intensive cooling at a speed of ˜2500 rpm. The brush was heldperpendicularly in gentle contact with the surfaces of the contaminatedsamples while moving in a circular motion.

The samples were initially brushed without paste for 1, 2 and 5 minutesin order to optimize the brushing time to that causing as little damageas possible to the surfaces (n=6 for each group). The implant-pasteformulations; a gel containing 10% (w/w) NMP and 20, 30, 50, or 60%(w/w) of hydrated silica in water were then assessed with the optimizedbrushing parameters (n=3 for each group). After that, the samples werebrushed with the optimized implant-paste and compared to surfacescleaned with rotary brushes alone and others brushed with a commercialtoothpaste (Colgate Total; Colgate-Palmoliven, New York, United states;n=6 for each group).

Analysis Methods

Ti surfaces were analyzed before and after the biofilm contamination andsubsequent brushing using the following methods:

X-Ray Photoelectron Spectroscopy (XPS)

XPS is the most widely used surface analysis technique that measures theelemental composition, chemical state and electronic state of theelements within a material. The chemical composition of Ti surfaces wasanalyzed using X-ray Photoelectron Spectrometer (Thermo FischerScientific Inc, East Grinstead, UK). The in was equipped with amonochromatic Al Ka X-Ray radiation source (1486.6 eV, (λ) 0.834 nm) andan ultrahigh vacuum chamber (10⁻⁹ torr). For all discs, survey scanswere acquired over the range of 0-1350 eV with a pass energy of 200 eVand a resolution of 1.0 eV. A flood gun was used to neutralize thesurface charging in all samples. Binding energies, peak areas and atomconcentration ratios were obtained using the curve fitting function ofAvantage (5.932v) analysis software (Thermo Fisher Scientific, Waltham,Mass. USA).

Live/Dead Bacterial Assays and Fluorescence Microscopy (FM)

Live/dead staining kit (BacLight Bacterial Viability Kit L7012,Molecular Probes, Carlsbad, USA) and fluorescence microscopy were usedto evaluate the viability and attachment of bacteria on the contaminatedand cleaned Ti discs (n=6 for each group). The live/dead stain wasprepared by diluting 1 L of SYTO 9 (excitation (λ)=485 nm, emission=498nm) and 1 μL of propidium iodide (excitation=535 nm, emission=617 nm) in1 mL of distilled water. Discs were placed in 48-well plate, and 500 μLof the reagent mixture was added to each well followed by incubation atroom temperature and in the dark for 15 min.

Each disc was then carefully placed on a glass slide, covered withmounting oil and stored in a dark space at 4° C. until furtherprocessing. Discs were evaluated using an upright fluorescencemicroscope (Carl Zeiss Microscopy GmbH, Gottingen, Germany) equippedwith a digital camera (AxioCam MRm Rev. 3, Carl Zeiss Microscopy,Gottingen, Germany) and operated with an image processing software (ZEN;Carl Zeiss Microscopy GmbH, Gottingen, Germany). For each disc, fiverandomly-selected sites were captured; one from the centre and the otherfour from the quarters of the Ti surface using a 20× objective. Means ofred fluorescent areas (dead cells), green fluorescent areas (viablecells), and total fluorescence (total bacteria) per standard microscopicfield area (448×335=0.15 mm²) were calculated (expressed as A.U.) usingCell Profiler image analysis software (Broad Institute of MIT andHarvard, Massachusettes, USA).

Scanning Electron Microscope (SEM)

Ti surfaces were scanned before and after biofilm contamination, andafter each cleaning procedure to visualize the surface contaminants orany topographical changes. Clean Ti discs were scanned with SEM (FE-SEMS-4700, Hitachi, Japan) without further preparation while thecontaminated discs were prepared as follows: the discs were fixed inglutaraldehyde (2.5% in phosphate buffered saline (PBS); PAALaboratories GmbH, Pasching, Austria) for 2 hours and washed 5 times for10 minutes in PBS, before dehydrating them in ascending concentrationsof ethanol (30-100 v/v %, 15 min each). The discs were then dried usingcritical point CO₂ (Ladd Research Critical Point Dryer). All discs weremounted on SEM-sample stubs and sputtered with gold. The SE mode with anacceleration voltage of 20 kV was selected, and the vacuum pressure wasmaintained below 1×10⁻⁵ torr. For direct comparison of surfacetopography, the same magnification of ×10,000 was selected for allsamples.

Confocal Laser Scanning Microscope

A LEXT 3D Confocal Microscope (Olympus America Inc., PA) was used toevaluate the surface roughness of polished Ti discs before and afterdecontamination. The surface roughness was characterized with roughnessprofile parameters [average roughness (Ra) and root mean squareroughness (Rq)]; a method extensively used for assessing the surfaceroughness of implants. All values were determined at a cut-off length of0.08 mm in 50 sections (evaluation length of 4 mm), and evaluated usingthe LEXT OLS4000 software (Olympus, America Inc., PA). Four discs wereused for each group, and measurements were taken at five random areasfrom each disc.

Statistical Analysis

The primary outcome variables were surface chemical composition, surfaceroughness, and bacterial attachment and viability. For each cleaningtechnique, data of the primary variables was statistically analyzedbased on paired design for comparison of the measurements from beforeand after contamination and decontamination. The outcomes of differentdecontamination methods were also analyzed and compared.

Repeated measures ANOVA and Paired-sample t-tests were used to comparethe outcomes of the same groups at different treatment time points whileone-way ANOVA and independent samples t-tests were performed to comparethe outcomes of different groups and techniques. The data analyses werecarried out using SPSS software version 22 (SPSS Inc., IBM Corporation,Somers, N.Y., USA) and Origin 9.0 (Origin lab, Northampton, Mass., USA).A p-value of <0.05 was set to represent a statistically significantdifference between groups.

Results Surface Chemistry of Clean and Biofilm-Contaminated Surfaces

The XPS survey spectra of clean surfaces showed the presence of thefollowing major peaks: O1s (Oxygen), C1s (Carbon), Ti2p (Titanium) andN1s (Nitrogen) (see FIGS. 60A and B). C1s and N1s signals indicate thesurface contamination while Ti2p signals demonstrate the presence of theTiO₂ oxide layer.

Biofilm contamination of Ti surfaces significantly increased C and Nlevels at the expense of 0 and Ti, indicating that the contaminants weremainly organic in nature (FIGS. 60A and B). Ti2p signal almostdisappeared from the spectra surveys of biofilm-contaminated surfacesindicating that the biofilm covers entirely the Ti surfaces with theorganic contaminants (FIG. 60A).

Note that the order of the columns in the bar chart in FIG. 60Bcorresponds to that set out in legend presented between (B) and (C).Also, in the bar chart, sa: significantly different from control (cleanTi) group, b: significantly different from biofilm-contaminated group,c: significantly different from Ti surfaces brushed for 1 minute, and d:significantly different from Ti surfaces brushed for 2 minutes (p<0.05).

Optimization of Brushing Time

Brushing Ti surfaces for 1 minute significantly decreased the levels ofC and N and increased the concentrations of O and Ti (FIG. 60B).Increasing the brushing time to 2 and 5 minutes did not achieve furthercleaning benefits but it induced surface scratches as seen on SEM images(FIG. 60C). Thus, the brushing time was fixed to 1 minute.

Optimization of Implant-Paste Formulation

The composition of the NMP gel was optimized as mentioned above toobtain an alkaline pH of 9.6. to 10% w/w.

The biofilm-contaminated surfaces were brushed using the optimized NMPgel (10% with respect to the water content) with and without hydratedsilica. SEM images showed that surfaces cleaned with the MNP gel and thegel with 30% hydrated silica were clean without noticeable changes intheir topography (see FIG. 61). Samples cleaned with the gel containing30% hydrated silica were significantly different from the contaminatedsamples in terms of elemental composition as shown by XPS, they showedhigher levels of Ti, 0 and lower levels of C, N, silica (Si) andmagnesium (Mg). The MNP gel with less silica also decreased C and Nlevels but they were less efficient than the gel containing 30% hydratedsilica. On the other hand, higher concentrations of silica (>30%) didnot improve the cleaning performance but caused surface contaminationwith implant-paste residues including Si. Small arrows in FIG. 61indicate the areas where the remnant silica accumulates on Ti surfaces.

This is also visible on FIG. 62. Note that the order of the columns inthe bar chart in FIG. 62 (from left to right) corresponds to that setout from top to bottom in the legend. Also, in the bar chart, a:significantly different from control group, b: significantly differentfrom biofilm contaminated group, c: significantly different from NMP gelgroup, d: significantly different from Ti surfaces brushed with the gelcontaining 20% hydrated silica, e: significantly different from Tisurfaces brushed with the gel containing 30% hydrated silica, and f:significantly different compared to Ti surfaces brushed with the gelcontaining 50% hydrated silica (p<0.05).

Based on these results, the optimized implant-paste formulation was theone containing 10% NMP gel and 30% hydrated silica.

Cleaning Uncontaminated (Control) Samples with the OptimizedImplant-Paste

Brushing uncontaminated Ti with the optimized implant-paste increasedthe surface levels of Ti while decreasing those of C (see FIG. 63). Inthis figure, a: significantly different from control group.

The commercial toothpaste did not show a change in Ti or C levels.

This indicates that the optimized implant-paste is also able to removethe carbon-containing compounds that are adsorbed from atmosphere andusually detected on clean Ti surfaces without inducing a significantincrease in their roughness as shown with confocal microscopy (see FIG.64). In this figure: *: significantly different from clean Ti surfaces,a: significantly different from Ti surfaces cleaned with the prophylaxisbrush, b: significantly different from Ti surfaces brushed withoptimized implant-paste (p<0.05).

The Optimized Implant-Paste Vs Colgate Toothpaste

The optimized implant-paste significantly reduced the atomicconcentration of surfaces' contaminants (C and N) and increased the Oand Ti levels. However, it did not induce any significant change in theTi surface roughness (see FIG. 64). Both results contrast with thoseobtained for surfaces cleaned with the brush alone or the brush withColgate toothpaste (FIGS. 65A and B). The toothpaste significantlyincreased the C levels and surface roughness of Ti.

Note that the order of the columns in the bar chart (from left to right)of FIG. 65 corresponds to that set out in legend (from top to bottom).Also, in the bar chart, a: significantly different from control group,b: significantly different from biofilm-contaminated group, c:significantly different from Ti surfaces cleaned with the prophylaxisbrush, d: significantly different from Ti surfaces brushed with theoptimized implant-paste (p<0.05).

The optimized implant-paste and Colgate toothpaste were able to removebacteria from biofilm-contaminated Ti surfaces reaching levels ofbacteria comparable to those found prior to biofilm contamination (FIGS.66A and B).

Note that the order of the columns in the bar chart (from left to right)of FIG. 66 corresponds to that set out in legend (1^(st) line and then2^(nd) line from left to right) presented between (A) and (B). Also, inthe bar chart, a: significantly different from control group, b:significantly different from biofilm-contaminated group, c:significantly different from Ti surfaces cleaned with the prophylaxisbrush, d: significantly different from Ti surfaces brushed with the gelcontaining 30% hydrated silica (p<0.05).

SEM images showed surfaces scratches and toothpaste residues on surfacescleaned with Colgate toothpaste but no scratches or residues could beseen with the optimized implant-paste (FIG. 61).

Discussion

Here we present a novel implant-paste specially designed and optimizedfor implant surface decontamination. It effectively disinfectscontaminated titanium implants without apparent negative impacts ontheir surface integrity.

In this study, we used in vivo biofilm model because it offers theopportunity to evaluate implant surfaces in realistic clinicalconditions; formation of composite plaque, co-adherence ofmicroorganisms and salivary pellicle under the removal forces ofsalivary flow and chewing activities 29. Several in vitro biofilm modelshave been tested and validated to study the implant surface bacterialinteractions 30-32. This includes for instance the commonly usedmicrotiter plate-based systems 33. However, they fail to preciselysimulate the complex structure of biofilm, the dynamics of itspathogenicity and ecological determinants 34, 35.

Prophylaxis instruments such as brushes and rubber cup are used toremove biofilms attached to implant surfaces with or without usingprophylaxis pastes. In this study, we used rotary brushes for cleaningTi surfaces because they are inexpensive and accessible compared totitanium brushes and their plastic bristles should be gentile on Ti.Rotating cups were found to leave remnants of rubber particles on theimplant surfaces after cleaning 5, 36. In addition, some cup materialsare too abrasive and can cause Ti surface damage 37.

In the present study, prophylaxis bushes were initially used todecontaminate the implant surfaces without a paste. The purpose of thisprocedure was to optimize the brushing time and exclude the possibledamaging effect of brushing technique. To the best of our knowledge,this is the first study that optimized the time required for Tidecontamination using the prophylaxis brush. The results showed thatbrushing the biofilm-contaminated implant surface for 1 minute was ableto remove the Ti surface contaminants efficiently (FIGS. 60 A and B),without inducing surface scratches or changes (FIGS. 60 C and D).

However, brushing for more than one minute induced visible scratches onthe Ti surfaces without improving the cleaning outcomes. Indeed,increasing the brushing time to 2 and 5 minutes negatively affects theTi surface topography and induced surfaces scratches (FIG. 60). Thisfinding could be attributed to the softness of Ti metal and its poorresistance to physical wear. Therefore, the brushes induced surfacescratches on Ti surface when brushing strokes have been increased morethan 2500 rpm. To confirm these results, the surface roughness ofpolished Ti surfaces was measured before and after the one-minutebrushing, which showed a significant increase in their roughness.However, the average surface roughness (Ra) after brushing was only0.018±0.004 μm, which falls within the reported threshold Ra (0.2 μm)that does not cause further alteration in surface microbiological load40.

Accordingly, brushing contaminated Ti implants surfaces for 1 minutes(2500 rpm) was able to decontaminate them without causing mechanicalabrasion, though the complete removal of contaminants andre-establishment of the Ti original chemistry were not achieved. Thisindicates the limited effectiveness of brushes in decontaminating Tisurfaces, which calls for the use of a dentifrice or a prophylaxispaste.

A dentifrice is usually combined with brushes to adjunct the physicalremoval of plaque and stains through their chemical and physicaladditives, or to apply therapeutic and preventive agents to toothsurfaces. The dentifrice needs two main ingredients to achieve themechanical cleaning; an abrasive agent and thickener to hold theabrasives in suspension during brushing. In this study, we developed andoptimized prophylaxis paste to decontaminate implant surfaces“implant-paste” and enhance the cleaning efficiency of the brush. Forthe development of this implant-paste, the sole thickener was composedof an inorganic, silicate free Nanocrystalline Magnesium Phosphate (NMP)gel. The gel composition was optimized to obtain an alkaline pH of 9.6because the corrosion resistance of Ti is high at this pH. In addition,the implant-paste can be in contact with intraoral structures and teethfor several hours when used for daily cleaning of Ti implants.Consequently, ideally, this optimized implant-paste to have a relativelyalkaline pH to minimize potential tooth or implant damage.

The optimized NMP gel has similar biocompatibility and thixotropicproperties of Laponite (silicate clays); the most used inorganicthickener in toothpastes. However, the optimized NMP gel has a stableconsistency without the need for additional organic thickeners. This isan advantage of our novel gel over the clays-based toothpastes thatrequire organic thickeners (i.e. xanthan gum) to provide optimalconsistency. The incorporated organic compounds could adhere to theimplant surfaces complicating their decontamination.

The other key component of the implant-paste that contributes to thephysical removal of biofilm is the abrasive agent. For ourimplant-paste, hydrated silica nanoparticles were chosen as abrasives.It is a relatively safe, nontoxic ingredient and mostly compatible withother ingredients, such as glycerine and fluoride. Moreover, lowconcentration of silicates shows osteoconductive properties that help toinduce and accelerate bone regeneration.

We used hydrated silica nanoparticles (˜200-300 nm) used as a polishingagent and optimized their content to obtain mild abrasiveness thatremoves plaque without scratching implant surfaces. It was also used toincrease the gel viscosity and to benefit from their osteoconductivityproperties. The study results showed that the best decontaminationoutcomes were obtained with the 10% NMP gel containing 30% hydratedsilica. This formula showed an efficient removal of the organiccontaminants from Ti surfaces and the least morphological/topographicalchanges (FIGS. 61 and 62).

The cleaning effectiveness of the optimized prophylaxis paste wasfurther confirmed by its ability to remove carbon-containing compoundfrom clean Ti surfaces (controls), using the optimized brushing time,without causing alteration in their roughness (FIG. 64). The significantdrop in carbon level (FIG. 63) after cleaning confirms the effectivenessof the implant-paste in removing the carbon-containing compound that arenormally adsorbed on Ti surface from atmosphere.

The superior decontamination efficiency of the implant-paste onbiofilm-contaminated surfaces in comparison to the rotary prophylaxisbrush and the brush with a commercial toothpaste was also observed (FIG.65). This confirms that the optimized implant-paste formulation is ableto remove the surface organic contaminants regardless of the mechanicalaction of the rotary brush. On the other hand, the significant increasein the carbon levels after cleaning these surfaces with Colgatetoothpaste indicates that the regular toothpastes further contaminatethe Ti surface. This result could owed to the organic content of thetoothpastes that are usually incorporated for thickening, binding orflavoring benefits. This confirms the superiority of the implant-pastedeveloped in this study, due to its inorganic nature, over the currentlyavailable pastes.

The bacteria attached to the surfaces brushed with the optimizedimplant-paste and Colgate toothpaste were found to be comparable to thatfound on uncontaminated Ti surfaces (FIG. 66). This result is inagreement with a previous study that indicated the superiority of atoothbrush and dentifrices over different ultrasonic scalers in reducingbacterial load from contaminated implant surfaces.

Conclusions

The optimized inorganic implant-paste shows superior efficiency indecontaminating implants than organic-based Colgate toothpaste withoutdamaging their surfaces integrity. The new inorganic implant-pastedeveloped in this study can remove biofilm from contaminated Ti implantswithout affecting their surface integrity. Cleaning dental implants withcurrent organic-based toothpastes contaminates the implants surfaces,changing their surface charge, roughness and chemistry, which could havenegative impact on re-osseointegration.

To the inventor's knowledge, this is the first paste ever speciallydesigned and optimized for implant surface decontamination. Theoptimized inorganic implant-paste shows 2 times more effective indecontaminating Ti, 3 times less abrasive than a regular toothpaste and2 times less bacteria than brushing alone. Therefore, it shows superiorefficiency in decontaminating Ti implants than organic-based toothpastewithout damaging their surfaces.

Example 4—Controlled Release of Drugs

Above, we showed that sodium magnesium phosphate nanocrystals (gel) areosteoinductive, thixotropic colloidal suspension and can be injectedthrough high gauge needles and therefore can be used for minimalinvasive interventions. Here, we investigated how this gel can controlthe release of local anesthetic (mepivacaine) in-vitro and in-vivo. Wealso investigated if the NMP loaded with mepivacaine could shortenpost-fracture mobilization time, reduce postoperative pain andaccelerate bone healing with the ultimate goal of developing aninjectable bone regeneration biomaterial with analgesic property.

Materials and Methods

The gel (NMP) for the in vitro release was prepared by dissolving 54 mgof Mg(OH)₂ (0.93 mmol, mole fraction 0.13) in 1.65 mL of H₃PO₄ 1.5 M(2.47 mmol, mole fraction 0.34), and subsequently 2.32 mL of NaOH 1.5 M(3.78 mmol, mole fraction 0.53) was added under vigorous stirring.

For the in vitro drug release, mepivacaine hydrochloride was dissolvedin 1 mL of gel previously prepared to reach different mepivacaineconcentration. The pH of the resulting thixotropic colloidal suspensionbefore the addition of mepivacaine was 7.95, while after the addition ofthe drug changed to 7.1. The gel+mepivacaine was placed in aPur-L-Lyzer™ dialysis tube with a molecular weight cut-off of 12000 Da,while the control group is represented by the mepivacaine dissolved inPBS (2% w/v) and placed in a dialysis tubes with the same molecularweight cut-off. The tubes were incubated in 30 mL of phosphate buffersaline solution (PBS) at 37° C., and the solution was changed atdifferent time points comprised between 0 and 168 hours. In order tocalculate the amount of drug released, the extracted solutions wereanalyzed with UV-Vis spectroscopy following the absorption ofmepivacaine at 263 nm. All experiments were done in triplicates.

The analgesic action of the mepivacaine with gel was assessed in vivousing the mouse-hindpaw-model. Twelve mice were assigned into fourgroups (n=3, each); saline, mepivacaine, gel and gel+mepivacaine. Eachmouse received a single injection of the assigned treatment (5 μLsubcutaneously) into the planter surface of the hindpaw. Due to thesmall volume injectable inside the hindpaw the amount of mepivacainedissolved inside the gel was increased from 2 to 8% w/v. This drugincrease drastically changed the pH of the final gel+mepivacainecolloidal dispersion from 7.5 to 4.1, being too acidic and subsequentlydestabilizing the gel structure provoking the precipitation of the gel.To address this problem, the gel for the in vivo experiment wassynthesized by dissolving 69 mg of Mg(OH)₂ (1.18 mmol, mole fraction0.16) in 1.65 mL of H₃PO₄ 1.5 M (2.47 mmol, mole fraction 0.31), andsubsequently 2.9 mL of NaOH 1.5 M (4.35 mmol, mole fraction 0.53) wasadded under vigorous stirring. For the in vivo drug release, 80 mg ofmepivacaine hydrochloride were dissolved in 1 mL of gel previouslyprepared (8% w/v). The pH of the resulting thixotropic colloidalsuspension before the addition of mepivacaine was 9.6, while after itsaddition the pH decreased to 7.6, being more compatible withphysiological pH. The sensitivity to thermal stimuli was tested usingradiant heat at different time points comprised between 0 and 120minutes.

The effects of NMP alone, NMP combined with mepivacaine and mepivacainealone, on post-fracture mobilization, postoperative pain and bonehealing were assessed in vivo using a standardized mice tibial fracturemodel (St-Arnaud, The Journal of steroid biochemistry and molecularbiology 121, 254-256 (2010)). Thirty-two mice were randomly assignedinto four groups (n=8, each); saline, mepivacaine, NMP andNMP+mepivacaine. Thirty minutes prior to surgical intervention, eachmouse was injected with Carprofen (20 mg/kg; SC).

The animal was then anesthetized with isoflurane (3-5% at the inductiontime and 2-2.5% during the maintenance period). After the animal showssigns of being fully anesthetized, the right leg was shaved anddisinfected using chlorohexidine, then, the animal was covered with asterile drape. A longitudinal skin incision was made in order to exposethe right patellar tendon. The tendon was dissected and elevated inorder to expose the proximal tibial tuberosity. A 27-gauge spinal needlewas introduced into the intramedullary canal of the tibia. A tibialfracture was performed in a standardized manner using a twister(Hiltunen et al., Journal of orthopaedic research 11, 305-312 (1993)).Following creation of fracture, a single post-operative injection of theassigned treatment (20 μL) into the fracture site and the surgical sitewas closed using 5-0 Vicryl.

Post-operative Animals' pain perception and locomotion were evaluated,at different time point; hours, 24 hours, 3 days, one week and two weekspost fracture, by assessing mice guarding (Yasuda et al., Journal ofpain research 6, 161 (2013)) and weight bearing per each leg,respectively. Animals were allowed to habituate to the testing room forat least one hour prior to handling. Guarding behavior was assessed asfollowing; each mouse was placed individually in a clear plastic box onan elevated stainless steel mesh. Both paws (of the fractured andnon-fractured legs) were observed closely for 1-minute periods everyfive minutes for 60 minutes. A score of 0, 1, or 2 was given accordingto the postural position of each paw in most of the 1-minute scoringperiods. If the injured side is blanched or distorted by the mesh, 0score was given. If the paw is completely off the floor, 2 score wasgiven. If the paw touches the floor without blanching or distorting, 1score was given. The sum of the 12 scores (one score every five minutesfor 60 minutes) (0-24) will be obtained during the 1-hour session foreach paw. The final guarding score was obtained by subtracting the scoreof the injured side from that of the non-injured hind paw (Yasada,supra).

Weight bearing test was performed as following; an incapacitance meter(IITC.inc, CA, US) was used for determination of hind paw weightdistribution. Mouse was placed in an angled plexiglass chamberpositioned so that each hind paw rest on a separate weighting plate. Theweight exerted by each hind limb was measured and averaged over a periodof 5 seconds. The change in hind paw weight distribution was calculatedby determining the difference in the amount of weight (g) between theleft and right limbs. Change in hind paw weight distribution between theleft (fractured leg) and right (control) limbs was used as an index ofpain in the fractured leg (Bove et al., Osteoarthritis and cartilage 11,821-830 (2003)). Two weeks following fracture, mice were euthanized andtibial explants were assessed for bone healing and fracture resistanceusing micro-CT and three point pending test, respectively.

Results and Discussion

The in vitro release experiment of mepivacaine demonstrates that,compared to the control formulation, the gel controlled the release ofmepivacaine.

As can be seen in FIG. 67, after 8 hours the release of mepivacaine inthe control group was almost complete with a total release of 90±5% ofdrug, while the group of gel+mepivacaine released 50±4% of the drug.Local anesthetics, such as mepivacaine and bupivacaine, have a shortduration effect, for example 2 hours. The release profile was slower forgel+mepivacaine than the control group indicating a prolonged releaseeffect, and thus a possible prolongation of the duration of thetherapeutic effect of mepivacaine.

The Korsmeyer-Peppa's model was used for the fitting of the cumulativedrug release as shown in FIG. 68. The equation of this model isM/M_(t)=kt^(n) where M/M_(t) is the cumulative amounts of drug releasedat time t, k is a constant incorporating structural and geometricalcharacteristics of the drug dosage form, and n is the release exponentthat identify the release mechanism. The linear fitting gave an exponentn=0.58 (R=0.99) and according to the Korsmeyer-Peppa's model when n is0.5<n<1 the mass transport is regulated by non-Fickian diffusion. Thisresult might indicate some physicochemical interaction between the drugand the gel which is responsible of the controlled released.

The stability of mepivacaine in contact with the gel was checked after24 hours from the beginning of the drug release experiment. The UV-Visspectra show the molecular integrity of the drug indicating thatmepivacaine is compatible with the gel (FIG. 69).

Radiant heat test showed that the gel loaded with mepivacaine providesanalgesia effect to mice and the analgesic action effect of the drug canbe prolonged using the gel (FIG. 70).

NMP combined with mepivacaine improved weight bearing on fractured legand reduced post-operative pain (FIG. 71).

NMP and NMP+mepivacaine accelerated fracture healing. Indeed, Micro-CTsagittal, coronal sections and 3 D reconstructions showed that boneformation at fracture site was higher in NMP alone and in NMP combinedwith mepivacaine (FIG. 72).

Force to fracture was significantly different between fractured andnon-fractured tibia, among saline, mepivacaine and NMP groups (FIG. 73).However, there was no significant difference between fractured andon-fractured legs among NMP+Mepivacaine group.

DISCUSSION AND CONCLUSION

The result of in vitro experiment showed that the gel is able to controlthe release of mepivacaine better than the control.

The biomaterial described here was injected through high gauge needleinto hindpaw of mice and provided effective analgesia to the micetreated with gel+mepivacaine.

In fact, the NMP was able to control the release of mepivacaine for upto 24 hours. Higuchi and Korsmeyer-Peppas models indicated thatmepivacaine was released by diffusion. The mepivacaine released by NMPpresented no change in its molecular structure as shown by UV-Visspectra (FIG. 69), indicating that the drug was compatible with NMP.Radiant heat test showed that NMP loaded with mepivacaine providesanalgesia and the analgesic action of mepivacaine was prolonged by NMP(FIG. 70). Guarding and weight bearing tests revealed that NMP loadedwith mepivacaine shorten the mobilization time and reduce postoperativepain (FIG. 71). Micro-CT data showed that NMP loaded with mepivacaine,accelerated bone healing (FIG. 72). Three point pending test indicatedthat NMP loaded with mepivacaine enhanced fracture resistance (FIG. 73).

The scope of the claims should not be limited by the preferredembodiments set forth in the examples, but should be given the broadestinterpretation consistent with the description as a whole.

REFERENCES

The present description refers to a number of documents, the content ofwhich is herein incorporated by reference in their entirety. Thesedocuments include, but are not limited to, the following:

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1. A pharmaceutical composition comprising one or more biactive agentsand a hydrogel as a carrier for the bioactive agent, wherein saidhydrogel comprises at least 1% v/v of a colloidal suspension of M^(I)_(X)M^(II) _(Y)P_(Z) two-dimensional nanocrystals in water, wherein:M^(I) is Na⁺ and/or Li⁺, M^(II) is Mg²⁺ or a mixture of Mg²⁺ with one ormore Ni²⁺, Zn²⁺, Cu²⁺, Fe²⁺ and/or Mn²⁺, P is a mixture of dibasicphosphate ions (HPO₄ ²⁻) and tribasic phosphate ions (P₄ ³⁻), X rangesfrom 0.43 to 0.63, Y ranges from 0.10 to 0.18, and Z ranges from 0.29 to0.48, X, Y, Z being mole fractions.
 2. The pharmaceutical composition ofclaim 1 being an implant.
 3. The pharmaceutical composition of claim 1being an injectable.
 4. The pharmaceutical composition of claim 1,wherein the bioactive agent is selected from the group consisting oflocal anaesthetic, antibiotics and beta blockers.
 5. A method oftargeting delivery of a bioactive agent to a site of need of a patient,the method comprising the steps of administering the pharmaceuticalcomposition of claim 1 to the site of need.
 6. The method of claim 5,wherein the site of need is a bone defect or a bone injury.
 7. Themethod of claim 5, wherein said administering step comprises implantingthe hydrogel.
 8. The method of claim 5, wherein said administering stepcomprises injecting the hydrogel.
 9. A paste for cleaning dentalimplant, the paste comprising a hydrogel mixed with an abrasive agent,wherein said hydrogel comprises at least 1% v/v of a colloidalsuspension of M^(I) _(X)M^(II) _(Y)P_(Z) two-dimensional nanocrystals inwater, wherein: M^(I) is Na⁺ and/or Li⁺, M^(II) is Mg²⁺ or a mixture ofMg²⁺ with one or more Ni²⁺, Zn²⁺, Cu²⁺, Fe²⁺ and/or Mn²⁺, P is a mixtureof dibasic phosphate ions (HPO₄ ²⁻) and tribasic phosphate ions (P₄ ³⁻),X ranges from 0.43 to 0.63, Y ranges from 0.10 to 0.18, and Z rangesfrom 0.29 to 0.48, X, Y, Z being mole fractions.
 10. The paste of claim9, wherein the hydrogel has a pH between about 9 and about
 10. 11. Thepaste of claim 9, wherein M^(I) is Na⁺, M^(II) is Mg²⁺, X is 0.0.50, Yis 0.13, and Z is 0.37.
 12. The paste of claim 9, wherein the abrasiveagent is a silica.
 13. The paste of claim 9, wherein the abrasive agentis hydrated silica nanoparticles.
 14. The paste of claim 9, whereinabrasive agent particles have a particles size up to about 500 nm. 15.The paste of claim 9, comprising from about 5 to about 60% by weight ofthe abrasive agent, based on the total weight of the paste.
 16. Thepaste of claim 9, further comprising one or more additives.
 17. Thepaste of claim 9, wherein the abrasive agent is selected from the groupconsisting of magnesium phosphate silica, a nano-silicate and calciumcarbonate.
 18. The paste of claim 9, wherein abrasive agent particleshave a particles size up to about 400 nm.
 19. The paste of claim 9,comprising from about 20% to about 40%, by weight of the abrasive agent,based on the total weight of the paste.